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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Bacterial Reverse Mutation: Under the conditions of this study, the test material was considered to be non-mutagenic.

Chromosome Aberration Test in Vitro: Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

Gene Mutation in Mammalian Cells: Under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.

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:
key study
Study period:
22 August 2012 - 22 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: guidelines published by the Japanese Regulatory Authorities, including METI, MHLW and MAFF.
Qualifier:
according to guideline
Guideline:
other: USA, EPA (TSCA) OPPTS harmonised guidelines.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine requirement in the Salmonella typhimurium strains.
Tryptophan requirement in the Escherichia 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):
- Type and identity of media: Stock cultures were prepared in Oxoid nutrient broth.
- Properly maintained: yes. Stored at approximately -196 °C in a liquid nitrogen freezer. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
Additional strain / cell type characteristics:
other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (10 % liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test
Experiment 1: 0, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
- EXPERIMENT 1
METHOD OF APPLICATION: in agar (direct plate incorporation)
0.1 mL aliquots of the bacterial cultures were dispensed into sets of test tubes, followed by 2 mL molten top agar (0.6 % agar, 0.5 % NaCl with 5 mL of 1.0 mM histidine and 1.0mM biotin for Salmonella typhimurium or 1.0 mM tryptophan solution for E. coli added to each 100 mL of top agar), 0.1 mL of the appropriate test material solution or the vehicle or positive control substance and 0.5 mL S9-mix (for the plates with metabolic activation) or 0.5 mL phosphate buffer (for the plates without metabolic activation). The contents were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
0.1 mL of the appropriate bacterial culture was dispensed into a test tube followed by 0.5 mL of S9 mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates.
The positive and untreated controls were dosed using the standard plate incorporation method described above.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The tests were performed in triplicate


DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All tester strain cultures should be in the range of 0.9 to 9 x 10⁹ bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA1537 (with and without S9 mix) at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA100 (without S9-mix only)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was performed with TA100 and WP2uvrA in both the absence and presence of S9-mix with ten different concentrations of the test material, ranging from 0.15 to 5000 µg/plate. The test material was not toxic at any concentration both in the absence and presence of S9-mix.


DEFINITIVE STUDY
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The mean number of revertant colonies for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiments 1 and 2, respectively.

The test material caused a visible reduction in the growth of the bacterial background lawns of Salmonella typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods. No toxicity was noted to any of the remaining bacterial strains at any test material dose level in both experiments in either the absence or presence of S9-mix. The test material was therefore tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix employing each exposure method.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

All of the positive control chemicals induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

1

82

18

26

32

9

2

77

17

31

14

12

 

Table 2 Experiment 1

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

-

-

-

-

-

Solvent

50

150

500

1500

5000

91

82

85

85

80

70*

16

15

14

17

15

14

26

28

26

30

26

25

28

22

28

26

26

27

17

13

12

15

15

10*

+

+

+

+

+

+

Solvent

50

150

500

1500

5000

99

94

102

97

99

84

13

12

10

10

11

12

31

33

31

27

35

26

27

27

27

26

29

27

11

12

11

11

12

7*

                                                     Positive Controls

 

 

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

Mean no. colonies/plate

749

541

267

159

113

 

 

+

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

1281

261

264

176

217

*Sparse bacterial background lawn

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

 

Table 3 Experiment 2

+/- S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

-

-

-

-

-

-

-

Solvent

5

15

50

150

500

1500

5000

79

77

75

74

71

70

70

70*

17

19

11

21

15

14

16

15

27

24

24

29

26

25

27

29

15

13

13

13

14

11

11

16

12

11

7

10

8

10

10

7*

+

+

+

+

+

+

+

+

Solvent

5

15

50

150

500

1500

5000

85

85

74

82

84

85

76

72

12

12

12

12

10

10

10

11

26

26

27

24

31

24

23

29

26

23

15

20

20

18

18

19

11

8

8

12

9

8

8

6*

                                                     Positive Controls

 

 

-

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

Mean no. colonies/plate

514

590

484

141

714

 

 

+

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

Mean no. colonies/plate

1455

246

200

223

281

*Sparse bacterial background lawn

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

Conclusions:
Interpretation of results: negative with and without metabolic activation.

Under the conditions of this study, the test material was considered to be non-mutagenic.
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. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at five and seven dose levels, respectively, both with and without metabolic activation. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate using the plate incorporation method and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate using the pre-incubation method.

The test material caused a visible reduction in the growth of the bacterial background lawns of S. typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods. No toxicity was noted to any of the remaining bacterial strains at any test item dose level in both experiments in either the absence or presence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. 

The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix.

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 August 2012 - 7 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The United Kingdom Environmental Mutagen Society (Cole et al, 1990)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell Culture: The stocks of cells were stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in Hams F12 medium, supplemented with 5 % foetal bovine serum (FBS) and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per mL) at 37 °C with 5 % CO₂ in air.

- Cell Cleansing: Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days (Hams F12 growth medium supplemented with Hypoxanthine (13.6 µg/mL, 100 µM), Aminopterin (0.0178 µg/mL, 0.4 µM) and Thymidine (3.85 µg/mL, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.
Metabolic activation:
with and without
Metabolic activation system:
Induced rat liver homogenate metabolising system (S9)
Test concentrations with justification for top dose:
Preliminary Cytotoxicity Test
- 0, 14.94, 29.88, 59.77, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL

Mutagenicity Test
- 4 hour exposure (-S9): 0, 239.06, 478.13, 956.25, 1912.5, 2868.75 and 3825 µg/mL
- 4 hour exposure (+2 % S9): 0, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL
- 24 hour exposure (-S9): 0, 29.88, 59.77, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL
- 4 hour exposure (+1 % S9): 0, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL
Vehicle / solvent:
Hams F12 culture medium. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12 culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.

MUTAGENICITY TEST
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. For the 4 hour exposure groups of Experiment 1, cells were seeded at 1.5 x 10⁶/75 cm² flask approximately 48 hours before being exposed to the test or control materials. In Experiment 2, cells were seeded approximately 48 hours before being exposed to the test or control materials at 1.0 x 10⁶/75 cm² flask for the 24 hour exposure group and at 1.5 x 10⁶/75 cm² flask for the 4 hour exposure group in the presence of S9.

Duplicate cultures were set up, both in the presence and absence of metabolic activation, with up to eight dose levels of test material, vehicle and positive controls. Treatment was for 4 hours in serum free media (Hams F12) or for 24 hours in Hams F12 with 1 % serum at 37 °C in an incubator with a humidified atmosphere of 5 % CO₂ in air.

At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in Hams F12 with 5 % FBS. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10⁶ cells/flask in a 225 cm² flask to allow growth and expression of induced mutants, and in triplicate in 25 cm² flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in Hams F12 with 5 % FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5 % CO₂ in air.

Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

During the 7 Day expression period the cultures were subcultured and maintained at 2 x 10⁶ cells/225 cm² flask on day 3 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:

i) In triplicate at 200 cells/25 cm² flask in 5 mL of Hams F12 with 5 % FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10⁵ cells/75 cm² flask (5 replicates per group) in Hams F12 with 5 % FBS, supplemented with 10 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37 °C in an incubator with humidified atmosphere of 5 % CO₂ in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.

The percentage of viability and mutation frequency per survivor were calculated for each dose group.

Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10 % Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.

i) The average absolute cloning efficiency of negative controls should be between 70 and 115 % with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70 % range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50 % cloning efficiency will be unacceptable.

ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10^-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10^-6 will not be used for the evaluation of a test material.

iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test material clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.

iv) Test materials with little or no mutagenic activity should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15 % of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test material in culture medium. Where a test material is excessively toxic, with a steep response curve, a concentration that is at least 75 % of the toxic dose level should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic.

v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.

vi) Five dose levels of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test material.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Experiment 1
A precipitate was seen at the end of exposure at and above 478.13 and 239.06 µg/mL in the absence and presence of S9 respectively.
There was no marked reduction in cloning efficiency demonstrated at Day 0 or Day 7 in the presence of S9. The 4 hour exposure group in the absence of S9 demonstrated an increase in toxicity at Day 0 to that seen in the Preliminary Cytotoxicity Test with a reduction in cloning efficiency of 73 % at the maximum dose when compared to the vehicle control group. There was also a reduction in the cloning efficiency at Day 7 in the absence of S9 at the maximum dose.
The Day 0 and Day 7 vehicle control cloning efficiencies for the 4 hour exposure group in the absence of S9 and the Day 0 cloning efficiencies for the 4 hour exposure group in the presence of S9 did not achieve 70 % in all the replicates; however since the value was greater than 50 % in all cases this was considered to be acceptable.
There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10^-6 in the presence of S9. In the 4 hour exposure in the absence of S9 the mutation frequency per survivor exceeded the vehicle control value by 20 x 10^-6 at 1912.5, 2868.75, and 3825 µg/mL. However, because the increases were within, or marginally above, the acceptable upper limit for a vehicle control the results were considered to be of no toxicological significance.
The vehicle control values were all within the maximum upper limit of 25 x 10^-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.


Experiment 2
A precipitate was seen at the end of the exposure period at and above 119.53 µg/mL in the presence of S9 and at and above 59.77 µg/mL in the absence of S9.
As in Experiment 1, there was no marked reduction in the Day 0 or Day 7 cloning efficiencies of the 4 hour exposure group in the presence of S9. The 24 hour exposure group demonstrated a dose related increase in toxicity at Day 0, similar to that seen in the Preliminary Cytotoxicity Test with a 70 and 62 % reduction in cloning efficiency at 1912.5 and 3825 µg/mL respectively when compared to the vehicle controls.
The Day 0 and the Day 7 vehicle control cloning efficiencies for the 24 hour exposure group did not achieve 70 % cloning efficiency in all the replicates; however since they achieved at least 50 % this was considered to be acceptable. Also, one of the replicates for the 4 hour exposure group in the presence of S9 at Day 0 did not achieve 70 % cloning efficiency but this was considered to be acceptable since it achieved at least 50 %.
There were no increases in mutation frequency per survivor that exceeded the vehicle control value by 20 x 10^-6 in either exposure group. The absence of a response in the 24 hour exposure group is considered to be confirmation that the response observed in the 4 hour exposure group in the absence of S9 in Experiment 1 did not have any toxicological significance.
It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10^-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Table 1 Experiment 1

Exposure Time (hours)

Dose

(µg/mL)

 

Day 7 Mutant

MF

MFS 10-6

SD

Group MFS 10-6

 

 

 

 

 

 

 

 

 

4 (-S9)

0

A

2

3.2

0.70

4

B

2

3.9

239.06

A

6

10.7

0.82

9

B

4

7.5

478.13*

A

6

10.5

0.70

13

B

8

14.8

956.25*

A

12

19.8

1.05

19

B

8

17.6

1912.5*

A

22

41.4

1.07

33

B

14

24.9

2868.75*

A

21

42.1

1.17

42

B

23

42.3

3825*

A

6

17.8

1.79

31

B

15

44.8

EMS 500

A

137

307.9

11.61

372

B

230

435.3

EMS 750

A

94

256.4

3.06

232

B

96

207.9

 

 

 

 

 

 

 

 

4 (+S9)

0

A

12

14.6

1.55

14

B

10

13.7

119.53

A

9

13.2

1.49

17

B

14

21.7

239.06*

A

6

9.4

1.71

18

B

18

26.7

478.13*

A

9

13.6

1.87

23

B

23

33.0

956.25*

A

12

17.1

1.40

13

B

6

9.0

1912.5*

A

7

10.8

1.89

14

B

13

16.8

3825*

A

14

18.2

1.32

18

B

14

17.7

DMBA 0.5

A

85

157.4

3.09

154

B

75

151.0

DMBA 1

A

132

313.0

6.18

261

B

98

208.5

*Precipitate

MF = Mutant frequency

MFS = Mutant frequency per survivor

SD = Standard deviation

EMS = Ethyl methane sulphonate

DMBA = Dimethyl benzanthracene

 

Table 2 Experiment 2

Exposure Time (hours)

Dose

(µg/mL)

 

Day 7 Mutant

MF

MFS 10-6

SD

Group MFS 10-6

 

 

 

 

 

 

 

 

 

24 (-S9)

0

A

0

0.0

0.32

1

B

1

1.5

29.88

A

7

7.8

1.03

5

B

1

1.6

59.77*

A

1

1.1

0.42

1

B

1

1.5

119.53*

A

7

8.1

1.03

5

B

1

1.5

239.06*

A

7

8.0

1.16

4

B

0

0.0

478.13*

A

9

9.6

0.79

7

B

3

4.3

956.25*

A

13

13.9

1.29

11

B

6

7.8

1912.5*

A

22

27.2

2.35

14

B

0

0.0

3825*

A

2

2.6

0.42

1

B

0

0.0

EMS 200

A

177

265.5

6.68

249

B

136

232.5

EMS 300

A

105

213.6

5.13

262

B

120

310.3

 

 

 

 

 

 

 

 

4 (+S9)

0

A

0

0.0

0.42

1

B

2

2.1

119.53*

A

2

3.4

0.70

4

B

4

4.4

239.06*

A

0

0.0

0.32

1

B

1

1.2

478.13*

A

1

1.5

0.42

1

B

1

1.2

956.25*

A

2

3.1

0.48

2

B

1

1.2

1912.5*

A

0

0.0

0.85

3

B

5

6.0

3825*

A

0

0.0

0.00

0

B

0

0.0

DMBA 0.5

A

22

63.8

2.77

50

B

19

36.9

DMBA 1

A

74

344.2

1.83

260

B

79

176.2

*Precipitate

MF = Mutant frequency

MFS = Mutant frequency per survivor

Conclusions:
Interpretation of results: negative with and without metabolic activation.

Under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.
Executive summary:

A study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells in vitro in accordance with the standardised guidelines OECD 476, EU Method B.17, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the EPA OPPTS 870.5300.

CHO cells were treated with the test material at up to eight dose levels, in duplicate, together with negative and positive controls. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.

Four treatment conditions were used for the test. In Experiment 1, a 4 hour exposure in the presence of 2 % S9 (dose levels 0, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL) and a 4 hour exposure in the absence of metabolic activation (dose levels 0, 239.06, 478.13, 956.25, 1912.5, 2868.75 and 3825 µg/mL). In Experiment 2, the 4 hour exposure was repeated using a 1 % final S9 concentration (dose levels 0, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL), whilst in the absence of metabolic activation the exposure time was increased to 24 hours (dose levels 0, 29.88, 59.77, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL).

In Experiment 1, a precipitate of the test material was seen at the end of exposure at and above 478.13 and 239.06 µg/mL in the absence and presence of S9, respectively. In Experiment 2, precipitate of the test material was seen at the end of the exposure period at and above 119.53 µg/mL in the presence of S9 and at and above 59.77 µg/mL in the absence of S9.

The 4 hour exposure group in the presence of S9 demonstrated no marked toxicity in any of the Experiments. The 4 hour exposure group in the absence of S9 demonstrated marked toxicity in Experiment 1 and the 24 hour exposure group also demonstrated a dose related increase in toxicity.

The test material demonstrated no dose related increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

The negative and positive controls gave mutant frequencies within the range expected, indicating the satisfactory performance of the test and of the metabolising system.

Under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2012 to 27 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

- Cell Culture
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air.
The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
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: 22.5, 45, 90, 180, 240, 360 µg/mL (with and without metabolic activation).
Experiment 2: 5.63, 11.25, 22.5, 45, 90, 180 µg/mL (without metabolic activation) and 22.5, 45, 90, 180, 240, 360 µg/mL (with metabolic activation).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Eagle's minimal essential medium with HEPES buffer (MEM)
- Justification for choice of solvent/vehicle: The test material was insoluble in dimethyl sulphoxide at 382.5 mg/mL but was suspended in aqueous media at 38.25 mg/mL. The culture medium was therefore selected as vehicle.

- Preparation of test material in vehicle
The test material was accurately weighed, dissolved in MEM and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into MEM and the osmolality did not increase by more than 50 mOsm.

The test material was formulated within two hours of it being applied to the test system.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Eagle's minimal essential medium with HEPES buffer (MEM)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Mitomycin C was dissolved in minimal essential medium and Cyclophosphamide was dissolved in DMSO
Details on test system and experimental conditions:
Culture Conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.94 – 9.15 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.65 - 0.75 mL heparinised whole blood

- With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at approximately 37 ºC, 5 % CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1 mL of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20 % S9¯mix (i.e. 2 % final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
In Experiment 2, 1 mL of 10 % S9-mix (i.e. 1 % final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.

- Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at approximately 37 ºC with 5 % CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1 mL of the appropriate vehicle control, test material solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1 mL of vehicle control, test material dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5 % CO2 in humidified air for 24 hours.

- Experiment 1
There were 2 exposure conditions conducted for Experiment 1:
i) 4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.

- Experiment 2
There were 2 exposure conditions conducted for Experiment 2:
i) 24-hour continuous exposure to the test material without S9-mix prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest.

- Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation.

- Slide Preparation
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

- Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material.

- Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

- Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50 % of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted. In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

- Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test material used was 14.94, 29.88, 59.77, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 μg/ml. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main test.
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 14.94 to 3825 μg/mL. The maximum dose was based on the 10 mM concentration. Precipitate observations were made from the blood-free cultures at the end of the exposure period. In the 4(20)-hour exposure group (without S9), precipitate was noted at the end of exposure at and above 239.06 μg/mL. However, in the 4(20)-hour exposure group (with S9), precipitate was noted at the end of exposure at all dose levels tested. In the 24-hour continuous exposure group, precipitate was observed at and above 119.53 μg/mL. Consequently, and due to the suspension of the test material, precipitate persisted on the slides at and above 239.06 μg/mL, which became obscuring at and above 956.25 μg/mL in the 4(20)-hour exposure groups. In the 24-hour continuous exposure group, this phenomenon was observed at and above 119.53 μg/mL and becoming obscuring at and above 956.25 μg/mL. No haemolysis was observed in any exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 3825 μg/mL in all three exposure groups. The selection of the maximum dose level was based on the onset of the precipitate in all exposure groups tested.

- Chromosome Aberration Test - Experiment 1
The qualitative assessment of the slides determined that the onset of precipitate was earlier in the absence of S9 and similar in the presence of S9 to that observed in the Preliminary Toxicity Test. There were metaphases suitable for scoring present up to the maximum dose level of 360 μg/mL in both in the presence and absence of S9. Precipitate observations were made at the end of exposure and precipitate in the pellet was noted at all dose levels tested in both exposure groups. Also, the precipitate persisted after exposure on the slides at and above 90 and 240 μg/mL in the absence and presence of S9, respectively.
The mitotic index data confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed.
The maximum dose level selected for metaphase analysis was 180 and 360 μg/mL in the absence and presence of S9, respectively, due to the presence of precipitate on the slides.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the presence or absence of metabolic activation.
The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

- Chromosome Aberration Test - Experiment 2
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 180 μg/mL in the absence of S9 and at 360 μg/mL in the presence of S9. Precipitate observations, made at the end of exposure, confirm that precipitate in the pellet was noted at all dose levels in both exposure groups and that the precipitate persisted after exposure on the slides at and above 90 and 180 μg/mL in the absence and presence of S9, respectively.
The mitotic index data show there was no dose-related reduction in Mitotic Index either in the absence or presence of S9.
The maximum dose level selected for metaphase analysis was therefore, 180 μg/mL and 240 μg/mL in the absence and presence of S9, respectively.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either exposure group.
The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Table 1: Mitotic Index Results

Dose level (µg/mL)

Experiment 1

Dose level (µg/mL)

Experiment 2

4 hr treatment without S9

4 hr treatment with S9

24 hr treatment without S9

4 hr treatment with S9

mean

% of control

mean

% of control

mean

% of control

mean

% of control

0

4.88

100

4.95

100

0

3.85

100

2.10

100

22.5

-*

-

-*

-

5.63

-*

-

NA

NA

45

4.30*

88

-*

-

11.25

-*

-

NA

NA

90

5.13*

105

-*

-

22.5

-*

-

-*

-

180

5.65*

116

4.75*

96

45

4.40*

114

-*

-

240

-*

-

3.85*

78

90

4.40*

114

2.63*

125

360

-*

-

4.00*

81

180

4.60*

119

2.48*

118

MMC 0.4

0.55

11

NA

NA

240

NA

NA

2.85*

136

CP 5

NA

NA

0.88

18

360

NA

NA

-*

-

MMC 0.2

1.70

44

NA

NA

CP 5

NA

NA

0.98

46

MMC = mitomycin C

CP = cyclophosphamide

NA = not applicable

- = not assessed for mitotic index

* precipitation observed

Table 2: Mean Results of Definitive Test

Treatment period (hours)

Conc. (µg/mL)

Mean Total % Cells with Structural Chromosome Aberrations (%)*

Gaps

Mean Total % of cells with Numerical Chromosome Aberrations (%)**

Experiment 1 without metabolic activation

4

Negative Control

0.0

0.0

0.0

45

0.0

0.0

0.0

90

1.0

0.5

0.0

180

0.0

0.5

0.0

MMC 0.4

44.0 (P< 0.001)

7.0

0.0

Experiment 1 with metabolic activation

4

Negative Control

1.5

1.0

0.5

180

0.5

1.5

0.0

240

1.0

0.0

0.0

360

2.0

0.5

0.0

CP 5

33.0 (P< 0.001)

5.0

1.0

Experiment 2 without metabolic activation

24

Negative Control

1.0

0.5

0.0

45

1.0

0.0

0.5

90

1.5

1.5

0.0

180

2.0

1.0

0.0

MMC 0.2

41.0 (P< 0.001)

7.0

0.0

Experiment 2 with metabolic activation

4

Negative Control

0.5

2.5

1.0

90

1.5

1.0

0.0

180

1.5

1.0

0.0

240

0.0

0.0

0.0

CP 5

38.0 (P < 0.001)

5.0

1.0

Results are the mean values from two replicates.

MMC = mitomycin; CP = Cyclophosphamide.

* Includes chromatid and chromosome breaks, chromatid and chromosome exchanges.

**Includes polyploids and other numerical chromosome aberrations.

Conclusions:
Interpretation of results:
negative with and without metabolic activation.

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.
Executive summary:

The potential of the test material to induce structural chromosomal aberrations was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 473 and EU Method B.10. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, including at least one precipitating concentration. Vehicle and positive controls were run concurrently. Four treatment conditions were used for the study. In Experiment 1, cultures were exposed for 4 hours with a 20 hour expression time, both in the presence and absence of metabolic activation (an induced rat liver homogenate at a final concentration of 2%). In Experiment 2, the 4 hour exposure period was repeated with metabolic activation (1 % final S9 concentration); whilst in the absence of metabolic activation cells were assayed with a continuous 24 hour exposure time. The frequencies of chromosome aberrations in both vehicle and positive controls were within the expected range and verified the sensitivity of the assay and the efficacy of the S9-mix.


Under the conditions of the test, exposure to the test material did not induce a dose-response reduction in mitotic indexes, or statistically significant increases in the frequency of structural chromosome aberrations, both in the presence and absence of metabolic activation. Furthermore, no polyploidy cells were recorded in any of the exposed cultures. In conclusion the test material was considered to be non-cytotoxic and non-clastogenic.

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

Additional information

In vitro data

Bacterial reverse mutation:

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. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997).

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at five and seven dose levels, respectively, both with and without metabolic activation. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate using the plate incorporation method and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate using the pre-incubation method. The test material caused a visible reduction in the growth of the bacterial background lawns of S. typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods. No signs of toxicity where displayed by any of the remaining bacterial strains, at any dose level with or without S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix. Under the conditions of the test, the test material was considered to be non-mutagenic in bacteria.

Chromosome aberration test in vitro:

The potential of the test material to induce structural chromosomal aberrations was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 473 and EU Method B.10. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, including at least one precipitating concentration. Vehicle and positive controls were run concurrently. Four treatment conditions were used for the study. In Experiment 1, cultures were exposed for 4 hours with a 20 hour expression time, both in the presence and absence of metabolic activation (an induced rat liver homogenate at a final concentration of 2%). In Experiment 2, the 4 hour exposure period was repeated with metabolic activation (1 % final S9 concentration); whilst in the absence of metabolic activation cells were assayed with a continuous 24 hour exposure time. The frequencies of chromosome aberrations in both vehicle and positive controls were within the expected range and verified the sensitivity of the assay and the efficacy of the S9-mix.
Under the conditions of the test, exposure to the test material did not induce a dose-response reduction in mitotic indexes, or statistically significant increases in the frequency of structural chromosome aberrations, both in the presence and absence of metabolic activation. Furthermore, no polyploidy cells were recorded in any of the exposed cultures. In conclusion the test material was considered to be non-cytotoxic and non-clastogenic.

Gene mutation in mammalian cells:

A study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells in vitro in accordance with the standardised guidelines OECD 476, EU Method B.17, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the EPA OPPTS 870.5300. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997).

During the study CHO cells were treated with the test material at up to eight dose levels, in duplicate, together with negative and positive controls. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.

Four treatment conditions were used for the test. In Experiment 1, a 4 hour exposure in the presence of 2 % S9 (dose levels 0, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL) and a 4 hour exposure in the absence of metabolic activation (dose levels 0, 239.06, 478.13, 956.25, 1912.5, 2868.75 and 3825 µg/mL). In Experiment 2, the 4 hour exposure was repeated using a 1 % final S9 concentration dose levels 0, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL), whilst in the absence of metabolic activation the exposure time was increased to 24 hours (dose levels 0, 29.88, 59.77, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL).

In Experiment 1, a precipitate of the test material was seen at the end of exposure at and above 478.13 and 239.06 µg/mL in the absence and presence of S9, respectively. In Experiment 2, precipitate of the test material was seen at the end of the exposure period at and above 119.53 µg/mL in the presence of S9 and at and above 59.77 µg/mL in the absence of S9.

The 4 hour exposure group in the presence of S9 demonstrated no marked toxicity in any of the Experiments. The 4 hour exposure group in the absence of S9 demonstrated marked toxicity in Experiment 1 and the 24 hour exposure group also demonstrated a dose related increase in toxicity. The test material demonstrated no dose related increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The negative and positive controls gave mutant frequencies within the range expected, indicating the satisfactory performance of the test and of the metabolising system. Therefore, under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.


Justification for selection of genetic toxicity endpoint
Multiple studies have been provided to address the different endpoint of genetic toxicity, each addressing different types of genetic toxicity. Since all the studies showed negative results, a single study could not be selected as key over the others.

Short description of key information:
In vitro Gene Mutation Study in Bacteria:
Ames: Harlan (2013), OECD 471 and EU Method B.13/14; Negative (with and without metabolic activation).

In vitro Mammalian Cell Cytogenicity:
Chromosome Aberration: Harlan (2013), OECD 473, EU Method B.10; Negative (with and without metabolic activation).

In vitro Gene Mutation Study in Mammalian Cells:
Chinese hamster Ovary (CHO): Harlan (2013), OECD 476, EU Method B.17, and EPA OPPTS 870.5300; Negative (with and without metabolic activation).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available genetic toxicity studies.