Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro gene mutation study in bacteria: The test item was determined to be non-mutagenic under the conditions of this test.

in vitro chromosome aberration study: The test item was considered to be non-clastogenic to human lymphocytes in vitro.

in vitro gene mutation study in mammalian cells: The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 22 October 2012 and 18 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10 % donor horse serum (giving R10 media) at 37 °C with 5 % CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.
Test concentrations with justification for top dose:
- Preliminary toxicity test: 3.91 to 1001.6 µg/mL
- Experiment 1 and 2: 0, 3.91, 7.83, 15.65, 31.3, 62.6, 125.2, 250.4, 500.8, 1001.6 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Sigma batch BCBH3157V at 400 μg/mL and 150 μg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
Acros batch A0302605 at 2 μg/mL was used as the positive control in the presence of metabolic activation for Experiments 1 and 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Preliminary toxicity test
- Exposure duration: 4 h with and without metabolic activation (S9); 24 h without S9
- Expression time (cells in growth medium): 24 h

Experiment 1
- Preincubation period:
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 10 to 14 d

Experiment 2
- Preincubation period:
- Exposure duration: 4 h with metabolic activation; 24 h without metabolic activation
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 10 to 14 d

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS)

NUMBER OF REPLICATIONS: The treatments were performed in duplicate (A + B), both with and without metabolic activation at 6 dose levels of the test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED: on day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL TFT in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (% V) in non-selective medium

DETERMINATION OF CYTOTOXICITY
- Method: The daily cell counts were used to obtain a Relative Suspension Growth (% RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (% V) data a Relative Total Growth (RTG) value.

OTHER
Preliminary toxicity test
- A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 h exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24 h exposure period without S9. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 10^5 cells/mL.
- The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 h by counting and diluting to 2 x 10^5 cells/mL. After a further 24 h the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (% RSG) value.
- Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20 % survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002)

Experiment 1
- Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at 6 dose levels of the test item (62.6 to 1001.6 μg/mL in both the absence and presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.
- The treatment vessels were incubated at 37 °C for 4 h with continuous shaking using an orbital shaker within an incubated hood.
- At the end of the treatment period the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and subcultured every 24 h for the expression period of 2 d, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.

Experiment 2
- An exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL cultures in R10 medium for the 4 h treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 h therefore 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm^2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (1 % S9 final concentration) at 6 dose levels of the test item (62.6 to 1001.6 μg/mL in both the absence and presence of metabolic activation), vehicle and positive controls. To each culture vessel was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24 h exposure group).
- The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 h in the absence of metabolic activation and 4 h in the presence of metabolic activation
- At the end of the treatment period the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and subcultured every 24 h for the expression period of 2 d, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
Evaluation criteria:
For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The IMF must exceed some value based on the global background MF for each method (agar or microwell). The Global Evaluation Factor (GEF) value is 126 x 10^-6 for the microwell method.

Any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 and demonstrate a positive linear trend will be considered positive.

If a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance.

When a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST: In the 4-hour exposure groups there was evidence of modest reductions in the Relative Suspension Growth (% RSG) of cells treated with the test item when compared to the concurrent vehicle controls. However, much greater reductions in % RSG were observed in the 24-hour exposure group and optimum levels of toxicity were achieved at the 10 mM limit dose level. Precipitate of the test item was not observed at any of the dose levels. Based on the % RSG values observed, the maximum dose level in the subsequent Mutagenicity Test was the 10 mM limit dose of 1001.6 μg/mL.

EXPERIMENT 1: There was evidence of modest dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and % RSG values. There was no evidence of any significant dose related reductions in viability (% V) in either the absence or presence of metabolic activation; therefore indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with both positive control substances.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, including the 10 mM limit dose level, in either the absence or presence of metabolic activation. Precipitate of the test item was not observed at any of the dose levels.

EXPERIMENT 2: There was evidence of modest toxicity following exposure to the test item in the 4-hour exposure group in the presence of metabolic activation, and more marked toxicity in the 24-hour exposure group in the absence of metabolic activation, as indicated by the % RSG and RTG values. There was no evidence of any significant dose related reductions in viability (% V) in either the absence or presence of metabolic activation; therefore indicating that residual toxicity had not occurred. Based on the % RSG and RTG values observed, optimum levels of toxicity were achieved in the 24-hour exposure group in the absence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances
The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item. The lowering of the S9 concentration to 1 % in this second experiment did not result in greater levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2 % metabolic activation in the Preliminary Toxicity Test and Experiment 1.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels in the presence of metabolic activation, including the 10 mM limit dose level. A very modest but statistically significant dose related (linear-trend) increase in mutant frequency was observed in the 24-hour exposure group in the absence of metabolic activation. However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels and the GEF was also not exceeded at any of the dose levels, including the 10 mM limit dose level that achieved optimum levels of toxicity. There was also no evidence of any significant increases in absolute numbers of mutant colonies and the mutant frequency values observed would have been considered acceptable for vehicle controls. The response was, therefore, considered to be artefactual and of no toxicological significance. Precipitate of the test item was not observed at any of the dose levels.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Preliminary toxicity test

The dose range of the test item used in the preliminary toxicity test was 3.91 to 1001.6 μg/mL. The results for the Relative Suspension Growth (% RSG) were as follows:

Dose (µg/mL)

% RSG (-S9)

4-Hour exposure

% RSG (+S9)

4-Hour exposure

% RSG (-S9)

24-Hour exposure

0

100

100

100

3.91

116

108

112

7.83

116

89

111

15.65

113

108

119

31.3

117

101

122

62.6

111

96

113

125.2

118

60

85

250.4

113

73

81

500.8

113

59

46

1001.6

88

79

12

Summary of Results

Experiment 1

4-Hour –S9

4-Hour +S9

Treatment (µg/mL)

% RSG

RTG

MF§

Treatment (µg/mL)

% RSG

RTG

MF§

0

100

1.00

157.56

0

100

1.00

128.80

62.6

89

1.10

108.68

62.6

93

1.00

154.20

125.2

102

1.04

131.97

125.2

91

1.09

127.08

250.5

87

1.02

132.37

250.5

83

0.96

113.87

500.8

74

0.89

116.84

500.8

82

0.96

154.48

751.2

86

0.87

131.97

751.2

88

0.93

153.47

1001.6

68

0.65

149.19

1001.6

68

0.82

148.99

Linear trend                                            NS

Linear trend                                     NS

EMS

400

76

0.58

1108.37

CP

2

63

0.42

1222.64

  

Experiment 2

4-Hour –S9

4-Hour +S9

Treatment (µg/mL)

% RSG

RTG

MF§

Treatment (µg/mL)

% RSG

RTG

MF§

0

100

1.00

120.30

0

100

1.00

122.50

62.6

88

0.87

103.16

62.6

89

1.03

108.50

125.2

84

0.89

129.14

125.2

86

0.90

103.50

250.5

71

1.02

118.93

250.5

80

0.72

130.32

500.8

31

0.45

132.83

500.8

83

0.96

120.77

751.2

14

0.26

130.88

751.2

83

0.85

123.40

1001.6

5

0.13

159.99

1001.6

75

0.74

144.28

Linear trend                                             *

Linear trend                                    NS

EMS

150

49

0.37

1388.93

CP

2

46

0.27

1028.72

% RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF§= 5- TFT resistant mutants/ 106 viable cells 2 days after treatment

NS= Not significant

* = p < 0.05

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

Conclusions:
The potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was assessed according to OECD guideline 476. The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

Introduction:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD guideline 476.

Methods:

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and for Experiments 1 and 2 was 62.6 to 1001.6 μg/ml in both the absence and presence of metabolic activation.

Results:

The maximum dose levels used in the Mutagenicity Test was the 10 mM limit dose of 1001.6 μg/mL. Precipitate of test item was not observed at any of the dose levels during the course of the study. The vehicle (DMSO) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at teh TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 March 2012- 23 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
No analysis was carried out to determine homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: histidine
E. coli: tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation test- experiment 1: 50, 150, 500, 1500 and 5000 µg/plate
Mutation test- experiment 2: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was fully miscible in DMSO at 50 mg/mL in solubility checks.
Untreated negative controls:
yes
Remarks:
spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
yes
Remarks:
spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Remarks:
spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION
Experiment 1: in agar (plate incorporation)
Experiment 2: preincubation

NUMBER OF REPLICATIONS: 3

PRELIMINARY TOXICITY TEST
A preliminary test was carried out to select the dose levels for the definitive tests. The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented , top agar, 0.1 mL of test item formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test item formulation and a vehicle control (DMSO) were tested. In addition, 0.1 mL of the maximum concentration of the test item and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertent colonies using a Domino colony counter and examined for effect on the growth of the bacterial background lawn.

MUTATION TEST-EXPERIMENT 1
Five concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Measured aliquots of 1 of the bacterial cultures were dispensed into sets of test tubes followed by 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL if the test item formulation, vehicle or positive control and either 0.5 mL or S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (1 tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.

All of the plates were incubated at 37 °C for approximately 48 h and the frequency of revertant colonies assessed using a Domino colony counter.

MUTATION TEST- EXPERIMENT 2
The second experiment was performed using fresh bacterial cultures, test item and control solutions.

Measured aliquots of 1 of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test item formulation and incubated for 20 min at 27 °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 in the surface of Vogel-Bonner Minimal agar plates (1 tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method.

All of the plates were incubated at 37 °C for approximately 48 h and the frequency of revertant colonies assessed using a Domino colony counter.
Evaluation criteria:
- A dose-related increase in mutant frequency over the dose range tested
- A reproducible increase at 1 or more concentrations
- Biological relevance against in-house historical control ranges
- Statistical analysis of data as determined by UKEMS
- Fold increase greater than 2 times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

Results for the negative controls (spontaneous mutation rates) were considered acceptable.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, reference item and vehicle controls, both with and without metabolic activation are attached

In the first experiment (plate incorporation methodology), the test item caused no visible reduction in the growth of the bacterial background lawns at any dose level either in the absence or presence of S9-mix. In the second experiment (pre-incubation method), the test item induced toxicity as weakened bacterial background lawns to all of the Salmonella strains dosed in the absence of S9-mix at 5000 µg/plate. No toxicity was noted in the Salmonella strains dosed in the presence of S9-mix or Escherichia coli strain WP2uvrA dosed in the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plated at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

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

Preliminary toxicity test. The number of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

 

 

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

137

126

122

123

128

127

133

111

109

107

129

+

TA100

103

128

138

109

112

115

114

104

93

126

96

-

WP2uvrA

21

27

26

28

19

25

19

22

20

32

18

+

WP2uvrA

37

31

30

18

37

25

22

21

29

26

25

Attachment 1: Table 2 Experiment 1- without metabolic activation

Attachment 2: Table 3 Experiment 1- with metabolic activation

Attachment 3: Table 4 Experiment 2- without metabolic activation

Attachment 4: Table 5 Experiment 2- with metabolic activation

Conclusions:
The mutagenic potential of the test item was assessed according to the OECD guideline 471. The test item was determined to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the OECD guideline 471.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at 5 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 50 - 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range (50 - 5000 µg/plate), fresh cultures of the bacterial strains and fresh test item formulations.

Results:

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All the positive controls used in the test induced marked increases in the frquency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

In the first experiment (plate incubation methodology), the test item caused no visible reduction in the growth of the bacterial background lawns at any dose level either in the absence or presence of S9-mix. In the second experiment (pre-incubation method), the test item induced toxicity as weakened bacterial background lawns to all of the Salmonella strains dosed in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to the Salmonella strains dosed in the presence of S9-mix or Escherichia coli strain WP2uvrA dosed in the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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

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

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2012- 20 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. certificate)
Remarks:
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
- Type and identity of media: 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 °C 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:
Induced rat liver homogenate metabolising system (S9)
Test concentrations with justification for top dose:
Preliminary test: 3.91, 7.83, 15.65, 31.3, 62.6, 125.6, 250.4, 500.8 and 1001.6 µg/mL
Experiment 1 and Experiment 2: 31.3, 62.6, 125.2, 250.4, 500.8 and 1001.6 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9, MMC was used at 0.4 and 0.2 µg/mL for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9, CP was used at 5 µg/mL in Experiment 1 and 2. It was dissolved in DMSO.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Preliminary Toxicity Test:
-Preincubation period, with and without metabolic activation: 48 h
- A preliminary toxicity test was performed on cell cultures using a 4-h exposure time with and without metabolic activation followed by a 20-h recovery period, and a continuous of 24 h with metabolic activation.

Experiment 1:
-Preincubation period, with and without metabolic activation: 48 h
- Exposure duration with and without S9-mix: 4 h
- Expression time (cells in growth medium) with and without S9-mix: 20-h culture in treatment-free media prior to cell harvest

Experiment 2:
- Preincubation period, with and without metabolic activation: 48 h
- Exposure duration without S9-mix: 24-h continuous
- Exposure duration with S9-mix: 4 h
- Expression time (cells in growth medium) with S9-mix: 20-h culture in treatment-free media prior to cell harvest

SPINDLE INHIBITOR: mitosis was arrested by addition of demecolcine 2 hr before the required harvest time
STAIN: when slides were dry they were stained in 5 % Giemsafor 5 min, raised, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS
Preliminary toxicity test:
- Parallel flasks, containing culture medium without whole blood, were established for the 3 exposure conditions so that the test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Experiment 1 and 2:
- Duplicate lymphocyte cultures (A and B) were established for each dose level

NUMBER OF CELLS EVALUATED: a total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as a mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Preliminary toxicity test: using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.
- 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 according to the simplified system of Savage (1976).

OTHER EXAMINATIONS:
- Determination of polyploidy: cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 1.

OTHER:
- cell harvest: After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075 M hypotonic KCI. After approximately 14 min (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCI cell suspension into fresh methanol/glacial acetic acid. The fixative was changed at least 3 times and the cells stored at approximately 4 °C to ensure complete fixation.
Evaluation criteria:
See Appendix 1: a positive response was recorded for a particular treatment if the % cells with aberrations, exclusing 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:
cytotoxicity
Remarks:
The test item only induced marked toxicity in the 24-h exposure group of Experiment 2 at the maximum recommended dose level.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period in any of the 3 exposure groups. Haemolysis was observed at the end of the exposure period in the 4(20)-h exposure groups at and above 250.4 µg/mL in the absence of S9 and at 1001.6 µg/mL in the presence of S9.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 101.6 in all 3 exposure groups. The mitotic data are presented in Table 1. The test item induced some evidence of toxicity in the 4(20)-h exposure group in the absence of S9 and in the 24-h exposure group.

The maximum dose level selected for all exposure groups of the main experiments was the maximum recommended dose level of 1001.6 µg/mL.

EXPERIMENT 1
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and there were metaphases suitable for scoring at the maximum recommended dose level, 1001.6 µg/mL. No precipitate of the test item was observed at the end of exposure in either exposure group.

The mitotic index data are given in Table 2. They confirm that qualitative observations in that no dose-related inhibition of mitotic index was observed, in either exposure group. Increases in the mitotic index over the vehicle control value may indicate some cell cycle delay which is indicative of toxicity which would corroborate the toxicity seen in the Preliminary Toxicity Test in the 4(20)-h exposure group in the absence of S9.

The maximum dose selected for metaphase analysis was the maximum recommended dose level (1001.6 µg/mL).

The chromosome aberration data are given in Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The response seen in the ‘B’ culture of the positive control in the 4(20)-h exposure group in the absence of S9 was initially weak and an extra 50 metaphases were therefore scored using a duplicate slide of better quality to verify the positive response. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The polyploidy cell frequency data are given in Tables 4 and 5. The test item did not induce a statistically significant increase in the numbers of polyploidy cells at any dose level in either ot the exposure groups.

EXPERIMENT 2
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 1001.6 µg/mL in both exposure groups. No precipitate of the test item was observed at the end of the exposure period in either exposure group.
The mitotic index data are given in Table 3. They confirm the qualitative observations in that a small dose-related inhibition of mitotic index was observed in the 4(20)-h exposure group in the presence of S9, and that 18 % mitotic inhibition was achieved at 1001.6 µg/mL. In the 24-h exposure group in the absence of S9 the toxicity was more marked with 40 % mitotic inhibition at 1001.6 µg/mL.

The maximum dose level selected for metaphase analysis was the maximum recommended dose level (1001.6 µg/mL).

The chromosome aberration data are given in Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

The polyploidy cell frequency data are given in Tables 6 and 7. The test item did not induce any statistically significant increases in the numbers of polyploidy cells at any dose level in either of the exposure groups.
Conclusions:
The cytogenicity potential of the test item was assessed according to OECD guideline 473. The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of the 2 separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it indentifies potential mutagens that produce chromosomal aberrations than gene mutations. The method used was designed to be compatible with that described in the OECD Guideline 473.

Methods:

Duplicate cultures of human lymphocytes, treated with the test item were evaluated for chromosome aberrations at 3 dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. In Experiment 1, 4 hr in the presence of an induced rat liver homogenate metabolising system (S9), at 2 % final concentration with cell harvest after a 20-hr expression period and a 4 hr exposure period in the absence of metabolic activation (S9) with a 20-hr expression period. In Experiment 2, the 4 hr exposure with addition of S9 was repeated (using a 1 % final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hr.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final concentration of test item (µg/mL)

4(20)-hr without S9

31.3, 62.6, 125.2, 250.4, 500.8, 1001.6

4(20)-hr with S9 (2 %)

31.3, 62.6, 125.2, 250.4, 500.8, 1001.6

24-hr without S9

31.3, 62.6, 125.2, 250.4, 500.8, 1001.6

4(20)-hr with S9 (1 %)

31.3, 62.6, 125.2, 250.4, 500.8, 1001.6

Results:

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items inducted statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of 2 separate experiments. The test item only induced marked toxicity in the 24-hr exposure group of Experiment 2 at the maximum recommended dose level.

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

Three in vitro genetic toxicity studies have been conducted on the test material, as follows:

in vitro gene mutation study in bacteria: The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the OECD guideline 471. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at 5 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 50- 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range (50- 5000 µg/plate), fresh cultures of the bacterial strains and fresh test item formulations. In the first experiment (plate incubation methodology), the test item caused no visible reduction in the growth of the bacterial background lawns at any dose level either in the absence or presence of S9-mix. In the second experiment (pre-incubation method), the test item induced toxicity as weakened bacterial background lawns to all of the Salmonella strains dosed in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to the Salmonella strains dosed in the presence of S9-mix or Escherichia coli strain WP2uvrA dosed in the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item was considered to be non-mutagenic under the conditions of the test.

in vitro chromosome aberration test: This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it indentifies potential mutagens that produce chromosomal aberrations than gene mutations. The method used was designed to be compatible with that described in the OECD Guideline 473. Duplicate cultures of human lymphocytes, treated with the test item were evaluated for chromosome aberrations at 3 dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. In Experiment 1, 4 hr in the presence of an induced rat liver homogenate metabolising system (S9), at 2 % final concentration with cell harvest after a 20-hr expression period and a 4 hr exposure period in the absence of metabolic activation (S9) with a 20-hr expression period. In Experiment 2, the 4 hr exposure with addition of S9 was repeated (using a 1 % final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hr.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final concentration of test item (µg/mL)

4(20)-hr without S9

31.3, 62.6, 125.2, 250.4, 500.8, 1001.6

4(20)-hr with S9 (2 %)

31.3, 62.6, 125.2, 250.4, 500.8, 1001.6

24-hr without S9

31.3, 62.6, 125.2, 250.4, 500.8, 1001.6

4(20)-hr with S9 (1 %)

31.3, 62.6, 125.2, 250.4, 500.8, 1001.6

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of 2 separate experiments. The test item only induced marked toxicity in the 24-hr exposure group of Experiment 2 at the maximum recommended dose level. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

in vitro gene mutation study in mammalian cells: The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD guideline 476. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation.The dose range of test item was selected following the results of a preliminary toxicity test and for Experiments 1 and 2 was 62.6 to 1001.6 μg/ml in both the absence and presence of metabolic activation.The maximum dose levels used in the Mutagenicity Test was the 10 mM limit dose of 1001.6 μg/mL. Precipitate of test item was not observed at any of the dose levels during the course of the study. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Justification for classification or non-classification

Three in vitro genetic toxicity studies have been conducted on the test material, an Ames study, a chromosome aberration study and a mouse lymphoma assay. All studies were conducted according to OECD guidelines and GLP and are adequately reported and therefore have been assigned a reliability 1.

The 3 in vitro genetic toxicity studies showed the test material to have no significant effects for genotoxicity. As such, the test material can be considered to be non-classified.