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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: Under the conditions of the study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay in both the presence and absence of metabolic activation.

Mouse Lymphoma Assay: Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Chromosome Aberation Assay: Under the conditions of this study, the test material is not considered to be a chromosome damaging (clastogenic) agent in vitro using human lymphocytes.

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
Study period:
11 July 2016 to 22 August 2016
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
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
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
No correction factor required.
Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid nutrient broth) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid nutrient broth) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196°C).
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver S9-mix induced by Aroclor 1254)
Test concentrations with justification for top dose:
- Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 0.54, 1.7, 5.4, 17, 52 and 164 μg/plate (absence of S9-mix) and 1.7, 5.4, 17, 52, 164 and 275 μg/plate (presence of S9-mix)
- Experiment 1A: TA1535, TA1537 and TA98 5.4, 17, 52, 164 and 512 µg/plate (absence of S9-mix) and TA98 5.4, 17, 52, 164 and 512 µg/plate (presence of S9-mix)
- Experiment 2 (all strains): 15, 27, 48, 86, 154 and 275 μg/plate (absence and presence of S9-mix)
- Experiment 2A (TA 98 and WP2uvrA): 17, 52, 164, 512 and 1000 µg/plate (presence of S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Chosen following a solubility test
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DOSE RANGE FINDING TEST/ MUTATION ASSAY
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix and reported as part of the first mutation experiment. The highest concentration of test material used in the subsequent mutation assay was the level at which the test material inhibited bacterial growth.

MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test material were tested in each strain both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
Statistics:
No formal hypothesis testing was done.
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST/FIRST MUTATION EXPERIMENT
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Dose range finding test: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix.
- First mutation experiment: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strains TA1535 and TA1537 in the presence of S9-mix. In strains TA1537 (absence of S9-mix) and TA98 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test material. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
- Mutagenicity: In tester strain TA1535, up to 5.8- and 3.0-fold increases were observed in the absence and presence of S9-mix, respectively. In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 1A
Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA1535, TA1537 and TA98 in the absence of S9- mix and TA98 in the presence of S9-mix, an additional experiment was performed.
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in all three tester strains.
- Mutagenicity: In tester strain TA1535, an up to 3.1-fold increase was observed in the absence of S9-mix. In the other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 2
To obtain more information about the possible mutagenicity of the test material, a second mutation experiment was performed in the absence and presence of 10 % (v/v) S9-mix.
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period.

- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strains TA98 and WP2uvrA in the presence of S9-mix. In strain TA98 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the lowest dose of 15 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test material. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.
- Mutagenicity: In the second mutation assay, no biologically relevant increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

MUTATION EXPERIMENT 2A
Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA98 and WP2uvrA in the presence of S9-mix, an additional experiment was performed.
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in both tester strains TA1535 and TA1537 in presence of S9-mix.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
In tester strain TA1535, increases in the number of revertant colonies more than three-times the concurrent vehicle control were observed. Although the increases observed in the absence of S9-mix were in two out of three experiments, the increases were not dose related, in the first experiment caused by one outlier and within the historical control data range, therefore these increases are considered to be not biologically relevant. The increase observed in the presence of S9-mix was in one experiment only, not dose related and within the historical control data range, therefore this increase is considered to be not biologically relevant.
The other four tester strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments performed.
The negative control values were within the laboratory historical control data ranges.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA100 in the second experiment (presence of S9-mix). In the second mutation assay, the mean plate count of the positive control of TA100 in the presence of S9-mix was not within the acceptability criteria. The value of 391 revertant colonies was outside the historical control data range. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.
Based on the results of this study it is concluded that the test material is not mutagenic.

Table 1: Dose Range-finder and Experiment 1 (Plate incorporation assay 5 % S9)

 

+/- S9 Mix

 

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Positive Control

Ethanol

0.54

1.7

5.4

17

52

164

512

1600

5000

1038

119

-

123

123

119

106

0 n

e MC

0

0

870

5

11

9

15

14

29

13 n NP

-

-

-

1751

29

-

19

20

17

17

9

2 n

0 a

0 NP a

1372

15

29

23

28

13

28

9 nNP

-

-

-

832

3

5

5

3

6

0

5 n NP

-

-

-

+

Positive Control

Ethanol

1.7

5.4

17

52

164

275

512

1600

5000

1264

124

140

142

137

95 n

17 s

-

0 e

0 a

0 NP a

241

9

15

11*

27

12

6 n

4 s NP

-

-

-

382

29

31

27

29

22

18

-

10

0 n

0 NP a

1176

20

8

14

27

21

23

15 n NP

-

-

-

443

7

15

6

7

5 n

0 s

E NP MC

-

-

-

Mean number of revertant colonies/3 replicate plates

MC = Microcolonies

NP = No precipitate

a = Bacterial background lawn absent

e = Bacterial background lawn extremely reduced

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

*Mean of two plates, one plate could not be determined due to a technical error

 

Table 2: Experiment 1A (Plate incorporation assay 5 % S9)

 

+/- S9 Mix

 

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Positive Control

Ethanol

5.4

17

52

164

512

-

-

-

-

-

-

-

934

9

12

27

28

12

0 n NP

-

-

-

-

-

-

-

1476

19

16

16

17

13

0 n NP

951

4

3

6

3

0

0 n NP

+

Positive Control

Ethanol

5.4

17

52

164

512

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1155

23

28

34

32

20

1 n NP

-

-

-

-

-

-

-

Mean number of revertant colonies/3 replicate plates

NP = No precipitate

n = Normal bacterial background lawn

 

Table 3: Experiment 2 (Plate incorporation assay 10 % S9)

 

+/- S9 Mix

 

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Positive Control

Ethanol

15

27

48

86

154

275

814

99

100

129

98

32 n

4 s

5 s NP

823

9

12

15

21

6

4 n

1 s NP

1277

18

29

18

16

16

13

3 n NP

1397

14

7

10

13

6

5

2 n NP

997

6

7

3

3 n

0 s

e MC

a NP

+

Positive Control

Ethanol

15

27

48

86

154

275

391

73

94

95

88

60 n

17 s

8 m NP

90

10

13

12

8

5

5

e NP MC

293

28

24

30

33

23

16

15 n NP

491

24

28

23

28

21

16

11 n NP

410

5

5

4 n

4 s

1 s

0 a

0 a NP

Mean number of revertant colonies/3 replicate plates

MC = Microcolonies

NP = No precipitate

a = Bacterial background lawn absent

e = Bacterial background lawn extremely reduced

m = Bacterial background lawn moderately reduced

n = Normal bacterial background lawn

s = Bacterial background lawn slightly reduced

 

Table 4: Experiment 2 (Plate incorporation assay 10 % S9)

 

+/- S9 Mix

 

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

Positive Control

Ethanol

17

52

164

512

1000

-

-

-

-

-

-

-

-

-

-

-

-

-

-

293

22

22

18

19

8

1 n NP

501

31

31

21

24

5

5 n NP

-

-

-

-

-

-

-

Mean number of revertant colonies/3 replicate plates

NP = No precipitate

n = Normal bacterial background lawn

Conclusions:
Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was evaluated in four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254) using ethanol as the vehicle. Additional experiments were performed to complete the data of the first and second experiment.

In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix. Results were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at the concentration ranges of 0.54 to 164 μg/plate in the absence of S9-mix and 1.7 to 275 μg/plate in the presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was only observed in TA1535 and TA1537 in the presence of S9-mix.

Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA1535, TA1537 and TA98 in the absence of S9- mix and TA98 in the presence of S9-mix, an additional experiment was performed (experiment 1A) in which the test material was tested at the concentration range of 5.4 to 512 μg/plate. Toxicity was observed in all tester strains.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 15 to 275 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all tester strains. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in the tester strains TA98 and WP2uvrA in the presence of S9-mix.

Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA98 and WP2uvrA in the presence of S9-mix, an additional experiment was performed (experiment 2A) in which the test material was tested at the concentration range of 17 to 1000 μg/plate. Toxicity was observed in both tester strains.

In tester strain TA1535, increases in the number of revertant colonies more than three-times the concurrent vehicle control were observed. Although the increases observed in the absence of S9-mix were in two out of three experiments, the increases were not dose related, in the first experiment caused by one outlier and within the historical control data range, therefore these increases are considered to be not biologically relevant. The increase observed in the presence of S9-mix was in one experiment only, not dose related and within the historical control data range, therefore this increase is considered to be not biologically relevant.

The other four tester strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments performed.

In this study, acceptable responses were obtained for the negative and strain-specific positive control materials indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 July 2016 to 03 October 2016
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 and 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro Mammalian Cell Gene Mutation Test
Target gene:
Induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection (ATCC, Manassas, USA) 2001

MEDIA USED
- Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
- Growth medium: Basic medium, supplemented with 10 % (v/v) heat-inactivated horse serum (= R10 medium). Horse serum was inactivated by incubation at 56 °C for at least 30 minutes.
- Exposure medium: For 3 hour exposure cells were exposed in basic medium supplemented with 5 % (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure cells were exposed in basic medium supplemented with 10 % (v/v) heat-inactivated horse serum (R10-medium).
- Selective medium: Basic medium supplemented with 20 % (v/v) heat-inactivated horse serum (total amount of serum = 20 %, R20) and 5 μg/mL trifluorothymidine (TFT).
- Non-selective medium: Basic medium supplemented with 20 % (v/v) heat-inactivated horse serum (total amount of serum = 20 %, R20).
- Properly maintained: Yes. Stock cultures of the cells were stored in liquid nitrogen (-196 °C). Cell density was kept below 1 x 10^6 cells/mL.
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. The cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
EXPERIMENT 1
3 hour treatment without S9-mix: 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 μg/mL exposure medium
3 hour treatment with S9-mix: 1, 5, 7.5, 10, 12, 13.5, 15, 17.5, 20, 22.5 and 25 μg/mL exposure medium

EXPERIMENT 2
24 hour treatment without S9-mix: 0.001, 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1 and 1.5 μg/mL exposure medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: No correction was made for the purity/composition of the test material. A solubility test was performed; the test material was dissolved in ethanol at a concentration of 50 mg/mL and formed a suspension at the concentration of 102.4 mg/mL and a clear solution at concentrations of 32.8 mg/ml and lower. The stock solution of 102.4 mg/mL was treated with ultrasonic waves to obtain a homogeneous suspension. The final concentration of the solvent in the exposure medium was 0.5 % (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
MUTAGENICITY TEST
All incubations were carried out in a humid atmosphere (80 - 100 %) containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C.
The test material was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods. Seven doses of the test material were tested in the mutation assay. Except in the first experiment in which six dose levels were tested in the presence of S9-mix.
The highest doses that were tested gave a cell survival of approximately 10-20 % and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

TREATMENT OF THE CELLS
Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes and incubated in a shaking incubator at 37.0 ± 1.0 °C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0 °C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to the test material in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test material in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed for to the test material in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and finally resuspension in 50 mL growth medium (R10).
For the 24 hour treatment, cell cultures were exposed to the test material in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were re-suspended in 20 mL growth medium (R10). The cells in the final suspension were counted with the coulter particle counter.

EXPRESSION PERIOD
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (where possible) were sub-cultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test material the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

DETERMINATION OF THE MUTATION FREQUENCY
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtitre plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells/concentration were plated in five 96-well microtitre plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtitre plates, each well containing 1000 cells in selective medium (TFT-selection). The microtitre plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) to each well. The plates for the CEday2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF MUTANT COLONIES
The colonies were divided into small and large colonies. The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

CALCULATION OF THE SURVIVAL OR VIABILITY
The suspension growth (SG) for the 3 hour treatment = [Day 1 cell count/1.6 x 10^5] x [Day 2 cell count/1.25 x 10^5]

The suspension growth (SG) for the 24 hour treatment = [Day 0 cell count/1.25 x 10^5] x [Day 1 cell count/1.25 x 10^5] x [Day 2 cell count/1.25 x 10^5]

Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100

The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells

The cloning efficiency (CE) was then calculated as follows:
CE = -ln P(0)/number of cells plated per well

The relative cloning efficiency (RCE) at the time of mutant selection = CE (test) / CE (controls) x 100

The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture:
RTG = RSG x RCE/100

CALCULATION OF THE MUTATION FREQUENCY
The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CEday2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CEday2 x 10^6

Small and large colony mutation frequencies were calculated in an identical manner.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met these criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120 % in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40 % of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control. The GEF has been defined as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test material is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGE FINDING TEST
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test material concentration range of 1.7 to 512 μg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
In the absence of S9-mix, the relative suspension growth was 25 % at the test material concentration of 5.4 μg/ml compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test material concentrations of 17 μg/ml and above. The test material precipitated in the exposure medium at the concentration of 164 μg/mL.
In the presence of S9-mix, the relative suspension growth was 35 % at the test material concentration of 17 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test material concentrations of 52 μg/mL and above. The test material precipitated in the exposure medium at the concentration of 164 μg/mL.
The relative suspension growth was 8 % at the test material concentration of 5.4 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test material concentrations of 17 μg/mL and above. The test material precipitated in the exposure medium at the concentration of 52 μg/mL.

EXPERIMENT 1
In the absence of S9-mix, the dose levels of 1 and 2 μg/mL, 3 and 4 μg/mL and 5 to 9 μg/mL showed similar cytotoxicity. Therefore, the dose levels of 2, 4, 6, 7 and 8 μg/mL were not regarded as relevant for mutation frequency measurement. The dose level of 10 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
In the presence of S9-mix, the dose levels of 10 and 12 μg/mL and 13.5 to 17.5 μg/mL showed similar cytotoxicity. Therefore, the dose levels of 12, 13.5 and 17.5 μg/mL were not regarded as relevant for mutation frequency measurement. The dose levels of 22.5 and 25 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.05, 0.1, 0.5, 1, 3, 5 and 9 μg/mL exposure medium
With S9-mix: 1, 5, 7.5, 10, 15 and 20 μg/mL exposure medium
In the absence of S9-mix, the relative total growth of the highest test material concentration was 11 % compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test material concentration was 18 % compared to the total growth of the solvent controls.

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
Increases above the 95 % upper control limit were observed at the dose levels of 5 and 9 μg/mL in the absence of S9-mix. Although these increases are above the 95 % upper control limit, the increases in the mutation frequency (164 and 148 per 10^6 survivors, for 5 and 9 μg/mL, respectively) are below the positive threshold of MF(controls) + 126 (225 x 10^-6) (Global evaluation factor, GEF) and not more than a 1.7-fold increase was observed, therefore these increases are considered to be not biologically relevant.

EXPERIMENT 2
The dose levels of 0.75 to 1.5 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.001, 0.005, 0.01, 0.05, 0.1, 0.25 and 0.5 μg/mL exposure medium.
The relative total growth of the highest test material was 8 % compared to the total growth of the solvent controls.

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test material. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

DISCUSSION
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95 % control limits of the distribution of the historical negative control database, except in the second experiment in which the mutation frequency of one of the solvent control cultures was not within the range of the acceptability criteria. In the second experiment, a total number of 178 wells was used in the solvent control (SC1) instead of 192 as specified in the protocol for determination of the CEday2. Due to a technical error, 14 wells could not be determined. Since the fall out was with a maximum of 8 %, the CEday2 could be determined, this fall-out had no effect on the results of the study.
Although the mutation frequency of one of the solvent control cultures in the first experiment observed in the presence of S9-mix was just above the upper control limits, these limits are 95 % control limits and a slightly higher response is within the expected response ranges.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The suspension growth over the two-day expression period for cultures treated with ethanol was between 12 and 16 (3 hour treatment) and 94 and 104 (24 hour treatment). In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Experiment 1: Cytotoxic and mutagenic response of the test material in the mouse lymphoma L5178Y test system

Dose (μg/mL)

RSG (%)

CEday2 (%)

RCE (%)

RTG (%)

Mutation frequency per 10^6 survivors

Total

Small

Large

Without metabolic activation - 3 hours of treatment

SC1

100

93

100

100

107

49

52

C2

89

90

54

33

0.05

110

71

79

87

112

58

50

0.1

95

83

91

87

107

67

36

0.5

42

105

116

49

76

27

46

1

34

120

132

45

87

40

42

3

23

107

118

27

97

41

52

5

19

77

85

16

164

72

82

9

14

68

75

11

148

74

67

MMS

95

55

60

57

822

363

367

With metabolic activation - 3 hours of treatment

SC1

100

91

100

100

132

67

58

SC2

83

162

106

47

1

95

71

82

78

112

42

66

5

43

91

105

45

121

69

45

7.5

41

97

111

46

102

58

39

10

38

69

80

30

121

84

32

15

24

104

119

29

81

54

24

20

20

77

88

18

106

67

35

CP

65

41

47

30

1814

1000

512

RSG = Relative Suspension Growth

CE = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

SC = Solvent control (ethanol)

MMS = Methylmethanesulfonate

CP = Cyclophosphamide

 

Experiment 2: Cytotoxic and mutagenic response of the test material in the mouse lymphoma L5178Y test system

Dose (μg/mL)

RSG (%)

CEday2 (%)

RCE (%)

RTG (%)

Mutation frequency per 10^6 survivors

Total

Small

Large

Without metabolic activation - 24 hours of treatment

SC1

100

85

100

100

67

17

48

C2

85

48

10

38

0.001

99

98

115

114

64

13

50

0.005

108

47

55

60

93

22

69

0.01

106

74

86

91

101

33

64

0.05

102

58

68

69

76

35

39

0.1

82

86

101

83

48

21

26

0.25

34

81

96

33

72

42

27

0.5

11

65

77

8

44

18

26

MMS

92

68

80

74

696

260

358

RSG = Relative Suspension Growth

CE = Cloning Efficiency

RCE = Relative Cloning Efficiency

RTG = Relative Total Growth

SC = Solvent control (ethanol)

MMS = Methylmethanesulfonate

Conclusions:
Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.
Executive summary:

The potential of the test material to cause mutagenic effects in mammalian cells was assessed in accordance with the standardised guideline OECD 490 under GLP conditions.

The mutagenicity evaluation of the test material was carried out in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. At concentrations of 102.4 mg/mL and higher the test material was suspended in ethanol and at concentrations of 32.8 mg/mL and lower the test material was dissolved in ethanol.

In the first experiment, the test material was tested up to concentrations of 9 and 20 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 11 and 18 % in the absence and presence of S9-mix, respectively. No precipitation was observed up to the concentration of 20 μg/mL.

In the second experiment, the test material was tested up to concentrations of 0.5 μg/mL in the absence of S9-mix. The incubation time was 24 hours and the RTG was 8 %. No precipitation was observed up to the concentration of 0.5 μg/mL.

The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay, except in the second experiment in which the mutation frequency of one of the solvent control cultures was not within the range of the acceptability criteria. Since the mutation frequency was just below the lower limit of the acceptability criteria range and the mutation frequency of the other solvent control culture was within the acceptability criteria range, this deviation in the mutation frequency had no effect on the validity of the results of the second mutation experiment.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The selection of the highest doses was based on the quality of the metaphases and not the mitotic index as specified in the guideline. The test material concentrations causing reduction of the mitotic index of about 50 % are at dose levels that severely affect the chromosomes, thus no longer allowing evaluation.
GLP compliance:
yes
Type of assay:
other: in vitro Mammalian Chromosome Aberration Test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
CELL CULTURE METHOD
"Chromosome medium 1A with PHA" (Gibco) was used as the culture medium; the total culture volume was 6.0 mL.
Human venous blood was drawn aseptically into sterile syringes that contained heparin to prevent clotting. About 0.5 mL of heparinised blood was added to 6.0 mL of culture medium in each centrifuge tube, and the cultures were incubated with closed caps at 37 °C.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix. A metabolic activation system using mouse livers was selected due to the fact that in a bioassay there was an incidence of hepatocellular tumours among mice but not among rats.
Test concentrations with justification for top dose:
0.125, 0.025, 0.05 µg/mL (without metabolic activation)
0.05, 0.10, 0.15 µg/mL (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Aqua dest. (distilled water)
Untreated negative controls:
yes
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:
METHOD OF APPLICATION: in medium
The volume of the blood culture was 6.8 mL consisting of the following components:
6.0 mL culture medium
0.5 mL heparinised blood
0.1 mL test material solution or control material
0.2 mL S-9 mix or phosphate buffer

- Assay without metabolic activation
Whole blood was added to the culture medium containing PHA and incubated at 37 °C for about 48 hours. Solutions of the test material, mitomycin C or the solvent and 0.2 mL phosphate buffer were then added to the cultures and subsequently re-incubated for further 24 hours (the whole duration of incubation was 72 hours). Colcemid was added 2 - 3 hours before harvesting the dividing lymphocytes.
- Assay with metabolic activation
Whole blood was added to the culture medium containing PHA and incubated at 37 °C for about 48 hours. Solutions of the test material, cyclophosphamide or the solvent and the S-9 mix were then added to the cultures. After about 2 hours of incubation at 37 °C, cells were washed twice with unsupplemented culture medium and then re-incubated in complete culture medium for further 22 hours. Colcemid was added 2 - 3 hours before fixation (the whole duration of incubation was 72 hours).

SPINDLE INHIBITOR (cytogenetic assays):
- Two to 3 hours prior to harvesting the cells 1.33 µg Colcemid/mL culture medium was added in order to arrest mitosis in the metaphase.

LYMPHOCYTE FIXATION AND CELL STAINING
After incubation at 37 °C, the blood cultures were centrifuged at 1000 rpm for 10 minutes, the supernatant was pipetted off except for a few drops, and the precipitate was re-suspended. After hypotonic treatment with about 9 - 10 mL of a hypotonlc solution (Hanks:aqua dest. 1:4) which was at 37 °C, the suspension was kept in a water bath at 37 °C for 20 minutes.
After re-centrifugation at 1000 rpm for 10 minutes, the supernatant was pipetted off except for one drop, and about 9 - 10 mL of fixative (methanol:glacial acetic acid 4:1) was slowly added to the sediment. After re-centrifugation at 1000 rpm the fixative was replaced, the centrifuge tube was closed with Parafilm and this suspension was kept at 4 °C for at least 10 minutes.
After re-centrifugation at 1000 rpm, the supernatant was pipetted off except for one drop, and a suspension was prepared with a few drops of fresh fixative. 3 - 5 drops of this suspension were dropped onto clean, shortly iced microscopic slides using a Pasteur pipette. The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol (10 mL Giemsa, 190 mL Titrisol pH 7.2) for 10 minutes.
After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Entellan.

NUMBER OF REPLICATIONS: Performed in duplicate

NUMBER OF CELLS EVALUATED: As a rule, 100 metaphases of each culture for the test material, negative and solvent controls or 50 cells of each culture for the positive controls were analysed for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. A mitotic index based on 1500 cells/culture was determined for at least the top two does that yield metaphase cells and for the positive and negative controls.

OTHER EXAMINATIONS:
- Determination of aneuploidy: Metaphases with absent (hypoploid) or additional (hyperploid) chromosomes. Only hyperploid metaphases were registered.
- Determination of polyploidy: Changes in the number of chromosomes by whole chromosome sets were determined.
Statistics:
The exact test according to Fisher, which is applied to register significant differences between the relative frequencies of a characteristic of two groups, was used to answer the questions of whether there are significant differences between control groups (untreated controls and solvent controls) and dose groups with regard to the rate of structural aberrant metaphases.
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ASSAY WITHOUT METABOLIC ACTIVATION
No differences regarding aneuploidies (hyperploid meta-phases) and polyploidies between the various dose groups and the negative controls were observed.
- Untreated control: 11 (5.5 %) aberrant cells incl. gaps and 1 (0.5 %) aberrant cell excl. gaps (1 x chromatid break) were found.
- Solvent control: 9 (4.5 %) aberrant metaphases incl. gaps and 2 (1.0 %) aberrant metaphases excl. gaps (2 x chromatid break) were observed.
- 0.05 µg/mL: 21 (10.5 %) chromosomally damaged cells incl. gaps and 4 (2.0 %) aberrant cells excl. gaps (1 x chromatid break, 1 x chromosome break and 2 x chromatid fragment) were detected.
- 0.025 µg/mL: 13 (6.5 %) aberrant metaphases incl. gaps and 3 (1.5 %) chromosomally damaged cells excl. gaps (1 x chromatid break, 1 x chromosome break and chromatid fragment and 1 x chromatid fragment) were observed.
- 0.0125 µg/mL: 14 (7.0 %) aberrant cells incl. gaps and 3 (1.5 %) aberrant metaphases excl. gaps (1 x chromosome deletion, 1 x chromosome fragment and 1 x exchange) were analysed.
- 0.2 µg mitomycin C/mL: With 62 (62 %) aberrant cells incl. gaps and 48 (48 %) aberrant mitosis excl. gaps including 4 multiple aberrant metaphases and 18 cells with exchanges, the positive control material led to the expected increase in the number of chromosomally damaged cells.

ASSAY WITH METABOLIC ACTIVATION
When compared to the negative control groups there were no differences regarding aneuploidies (hyperploid metaphases) and polyploidies in the dose groups.
- Untreated control: 9 (4.5 %) aberrant mitosis incl. gaps and 2 (1.0 %) aberrant cells excl. gaps (1 x chromatid fragment and chromosome fragment) were analysed.
- Solvent control: 9 (4.5 %) aberrant metaphases incl. gaps and 3 (1.5 %) chromosomally damaged cells excl. gaps (2 x chromatid breaks and 1 x 4 chromosome fragments) were found.
- 0.15 µg/mL: 23 (11.5 %) chromosomally damaged cells incl. gaps and 2 (1.0 %) aberrant cells excl. gaps (1 x chromatid break and 1 x chromosome break) were observed.
- 0.1 µg/mL: 25 (12.5 %) aberrant metaphases incl. only gaps were detected.
- 0.05 µg/mL: 15 (7.5 %) chromosomally damaged cells incl. gaps and 1 (0.5 %) aberrant cell excl. gaps (1 x chromatid break) were found.
- 6 µg cyclophosphamide/mL: With 44 (44 %) aberrant cells incl. gaps and 25 (25%) aberrant metaphases excl. gaps including 6 cells with exchanges, the positive control material led to the expected increase in the number of chromosomally damaged cells.

DISCUSSION
According to the results of the study, the test material caused a slight increase in the number of aberrant metaphases but only including gaps without S-9 mix at 0.05 µg/ml (statistically not significant) or with S-9 mix at 0.15 µg/ml (statistically not significant) and at 0.1 µg/ml (statistical significance of 95 %). However, the genetic consequences of gaps are rather uncertain and the occurrence of this aberration type alone is no suitable criteria for the evaluation of a clastogenic event.

Table 1: Results of Chromosome Aberration Test (24 hours after administration)

Experiment without metabolic activation

 

Untreated Control

Vehicle Control

0.05

µg/mL

0.025

µg/mL

0.0125

µg/mL

MMC 0.2

µg/mL

Culture total

2

2

2

2

2

2

Analysed metaphases

200

200

200

200

200

100

Number of cultures with aberrant metaphases

2

2

2

2

2

2

Metaphases with aberrations

Including gaps / 100

11 / 5.50

9 / 4.50

21 / 10.50

13 / 6.50

14 / 7.00

62 / 62.00

xx++

Excluding gaps

1 / 0.50

2 / 1.00

4 / 2.00

3 / 1.50

3 / 1.50

48 / 48.00

xx++

Exchanges

0 / 0.0

0 / 0.0

0 / 0.0

0 / 0.0

1 / 0.50

18 / 18.00

xx++

Mult. Aber. Met

0

0

0

0

0

4

Pulverisations

0

0

0

0

0

0

Aneuploidy metaphases

0 / 0.0

0 / 0.0

0 / 0.0

1 / 0.50

0 / 0.0

0 / 0.0

Polyploidy metaphases

1 / 0.50

1 / 0.50

0 / 0.0

0 / 0.0

3 / 0.50

0 / 0.0

Experiment with metabolic activation

 

Untreated Control

Vehicle Control

0.15

µg/mL

0.1

µg/mL

0.05

µg/mL

CPP

6 µg/mL

Culture total

2

2

2

2

2

2

Analysed metaphases

200

200

200

200

200

100

Number of cultures with aberrant metaphases

2

2

2

2

2

2

Metaphases with aberrations

Including gaps / 100

9 / 4.50

9 / 4.50

23 / 11.50

25 / 12.50

15 / 7.50

44 / 44.00 xx++

Excluding gaps

2 / 1.00

3 / 1.50

2 / 1.00

0 / 0.0

1 / 0.50

25 / 25.00 xx++

Exchanges

0 / 0.0

0 / 0.0

0 / 0.0

0 / 0.0

0 / 0.0

6 / 6.00 xx++

Mult. Aber. Met

0

0

0

0

0

0

Pulverisations

0

0

0

0

0

0

Aneuploidy metaphases

0 / 0.0

0 / 0.0

1 / 0.50

2 / 1.00

1 / 0.50

0 / 0.0

Polyploidy metaphases

0 / 0.0

1 / 0.50

2 / 1.00

1 / 0.50

1 / 0.50

0 / 0.0

MMC = Mitomycin C

CPP = Cyclophosphamide

A significant increase in the number of aberrant cells against the untreated controls and solvent controls at the 95 % level is marked + and x respectively; an increase at the 99 % level is marked ++ and xx respectively.

Table 2: Determination of Mitotic Index (%)

Test Group (µg/mL)

S-9 Mix

1st Culture (%abs.)

1st Culture (%abs.)

Mean (% abs.)

% Rel.

Untreated control

-

5.9

5.5

5.7

100

Solvent control

6.2

7.0

6.6

100

0.05

5.7

5.0

5.4

81.8

0.025

7.1

5.1

6.1

92.4

0.0125

5.7

5.9

5.8

87.9

Positive control

3.3

3.1

3.2

48.5

Untreated control

+

7.4

6.5

7.0

100

Solvent control

9.1

6.8

8.0

100

0.15

5.7

8.6

7.2

90.0

0.1

9.1

9.3

9.2

115.0

0.05

6.5

9.8

8.2

102.5

Positive control

5.7

6.7

6.2

77.5

Conclusions:
Under the conditions of this study, the test material is not considered to be a chromosome damaging (clastogenic) agent in vitro using human lymphocytes.
Executive summary:

The potential of the test material to induce chromosome aberrations in human lymphocytes was assessed in accordance with the standardised guideline OECD 473 under GLP conditions.

The test material was evaluated in vitro in both the absence and presence of a metabolising system (S9 -mix derived from mice). A pre-test was conducted for the determination of the highest experimental dose and due to cytotoxicity of the test material, 0.05, 0.025 and 0.0125 µg/mL culture medium was used in the experiment without S-9 mix and 0.15, 0.10 and 0.05 µg/mL culture medium was used in the experiment with metabolic activation. This selection was based on the quality of the metaphases and not on the mitotic index, because the test material concentrations causing a reduction of the mitotic index were at dose levels that severely affect chromosomes, thus no longer allowing evaluation.

The various doses were dissolved in aqua dest. For control purposes, negative controls (untreated and solvent) and positive controls both without S-9 mix (0.2 µg mitomycin C/mL culture medium) and with metabolic activation (6 µg cyclophosphamide/mL culture medium) were also tested. Duplicate cultures were used for all experimental points.

Heparinised human venous blood was added to the culture medium (chromosome medium 1A with PHA). After mitogen stimulation of the lymphocytes using PHA and incubation at 37 °C for 48 hours, the cultures were treated with the test material in the experiment without S-9 mix for 24 hours; in the experiment with S-9 mix, test material treatment lasted 2 hours followed by a re-incubation for 22 hours using fresh culture medium without test material. About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells in a metaphase-like stage of mitosis (C-metaphase). After preparation of the lymphocyte chromosomes and staining with Giemsa, 100 metaphases of each culture in the case of the test material, untreated control and solvent control, or 50 cells of each culture in the case of the positive controls, were analysed for chromosomal aberrations.

 According to the results of the present study, the test material did not lead to a biologically significant increase in the number of aberrant metaphases with or without S-9 mix.

Under the conditions of this study, the test material is not considered to be a chromosome damaging (clastogenic) agent in vitro using human lymphocytes.

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

Additional information

The following studies were assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Ames Test

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was evaluated in four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254) using ethanol as the vehicle. Additional experiments were performed to complete the data of the first and second experiment.

In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix. Results were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test material was tested in the first mutation assay at the concentration ranges of 0.54 to 164 μg/plate in the absence of S9-mix and 1.7 to 275 μg/plate in the presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was only observed in TA1535 and TA1537 in the presence of S9-mix.

Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA1535, TA1537 and TA98 in the absence of S9- mix and TA98 in the presence of S9-mix, an additional experiment was performed (experiment 1A) in which the test material was tested at the concentration range of 5.4 to 512 μg/plate. Toxicity was observed in all tester strains.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 15 to 275 μg/plate in the absence and presence of 10 % (v/v) S9-mix in all tester strains. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in the tester strains TA98 and WP2uvrA in the presence of S9-mix.

Since insufficient toxicity without precipitate on the plates was observed in the tester strains TA98 and WP2uvrA in the presence of S9-mix, an additional experiment was performed (experiment 2A) in which the test material was tested at the concentration range of 17 to 1000 μg/plate. Toxicity was observed in both tester strains.

In tester strain TA1535, increases in the number of revertant colonies more than three-times the concurrent vehicle control were observed. Although the increases observed in the absence of S9-mix were in two out of three experiments, the increases were not dose related, in the first experiment caused by one outlier and within the historical control data range, therefore these increases are considered to be not biologically relevant. The increase observed in the presence of S9-mix was in one experiment only, not dose related and within the historical control data range, therefore this increase is considered to be not biologically relevant.

The other four tester strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in any of the experiments performed.

In this study, acceptable responses were obtained for the negative and strain-specific positive control materials indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Mouse Lymphoma Assay

The potential of the test material to cause mutagenic effects in mammalian cells was assessed in accordance with the standardised guideline OECD 490 under GLP conditions.

The mutagenicity evaluation of the test material was carried out in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. At concentrations of 102.4 mg/mL and higher the test material was suspended in ethanol and at concentrations of 32.8 mg/mL and lower the test material was dissolved in ethanol.

In the first experiment, the test material was tested up to concentrations of 9 and 20 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was 11 and 18 % in the absence and presence of S9-mix, respectively. No precipitation was observed up to the concentration of 20 μg/mL.

In the second experiment, the test material was tested up to concentrations of 0.5 μg/mL in the absence of S9-mix. The incubation time was 24 hours and the RTG was 8 %. No precipitation was observed up to the concentration of 0.5 μg/mL.

The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay, except in the second experiment in which the mutation frequency of one of the solvent control cultures was not within the range of the acceptability criteria. Since the mutation frequency was just below the lower limit of the acceptability criteria range and the mutation frequency of the other solvent control culture was within the acceptability criteria range, this deviation in the mutation frequency had no effect on the validity of the results of the second mutation experiment.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency.

Under the conditions of this study, it is concluded that the test material is not mutagenic in the TK mutation test system.

Chromosome Aberration

The potential of the test material to induce chromosome aberrations in human lymphocytes was assessed in accordance with the standardised guideline OECD 473 under GLP conditions.

The test material was evaluated in vitro in both the absence and presence of a metabolising system (S9 -mix derived from mice). A pre-test was conducted for the determination of the highest experimental dose and due to cytotoxicity of the test material, 0.05, 0.025 and 0.0125 µg/mL culture medium was used in the experiment without S-9 mix and 0.15, 0.10 and 0.05 µg/mL culture medium was used in the experiment with metabolic activation. This selection was based on the quality of the metaphases and not on the mitotic index, because the test material concentrations causing a reduction of the mitotic index were at dose levels that severely affect chromosomes, thus no longer allowing evaluation.

The various doses were dissolved in aqua dest. For control purposes, negative controls (untreated and solvent) and positive controls both without S-9 mix (0.2 µg mitomycin C/mL culture medium) and with metabolic activation (6 µg cyclophosphamide/mL culture medium) were also tested. Duplicate cultures were used for all experimental points.

Heparinised human venous blood was added to the culture medium (chromosome medium 1A with PHA). After mitogen stimulation of the lymphocytes using PHA and incubation at 37 °C for 48 hours, the cultures were treated with the test material in the experiment without S-9 mix for 24 hours; in the experiment with S-9 mix, test material treatment lasted 2 hours followed by a re-incubation for 22 hours using fresh culture medium without test material. About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells in a metaphase-like stage of mitosis (C-metaphase). After preparation of the lymphocyte chromosomes and staining with Giemsa, 100 metaphases of each culture in the case of the test material, untreated control and solvent control, or 50 cells of each culture in the case of the positive controls, were analysed for chromosomal aberrations.

 According to the results of the present study, the test material did not lead to a biologically significant increase in the number of aberrant metaphases with or without S-9 mix.

Under the conditions of this study, the test material is not considered to be a chromosome damaging (clastogenic) agent in vitro using human lymphocytes.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.