Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was assessed for genetic toxicity in three in vitro studies. An in vitro gene mutation study in bacteria was performed according to OECD Test Guideline 471 using a plate incorporation test and a pre-incubation test with and without metabolic activation. The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. An in vitro cytogenicity study in mammalian cells was conducted according to OECD Test Guideline 473. The method was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The test item did not induce any toxicologically significant increases in the mutant frequency An in vitro gene mutation study in mammalian cells was conducted according to OECD Test Guideline 476 for the detection of structural chromosomal aberrations in cultured mammalian cells. The test substance was non-clastogenic to human lymphocytes in vitro.

Link to relevant study records

Referenceopen allclose all

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:
The study was conducted between 09 May 2014 and 25 June 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
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: Kanpoan No. 287 - Japanese Environment Protection Agency; Eisei No. 127 - Japanese Ministry of Health and Welfare and Heisei 09/10/31 Kikyoku No. 2 - Japanese Ministry of International Trade & Industry.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Batch: SC00010054
Purity: 96.5%
Physical state/Appearance: Clear colourless liquid
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1
4-hour without S9: 2.5, 5, 10, 20 µg/mL
4-hour with S9 (2%): 2.5, 5, 10, 20, 40, 50 µg/mL

Experiment 2
24-hour without S9: 2, 4, 8, 16, 24, 32 µg/mL
4-hour with S9 (2%): 8, 16, 32, 40, 48, 56 µg/mL
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. 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. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Mutagenicity Test
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 1E+06 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 eight dose levels of the test item (2.5 to 40 µg/mL in the absence of metabolic activation and 2.5 to 70 µg/mL in the 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 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL cultures in R10 medium for the 4 hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3E+06 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (2 to 48 µg/mL in the absence of metabolic activation, and 2 to 56 µg/mL in the 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 controls) and sufficient R0 medium to give a final volume of 20 mL (R10 was used for the 24 hour exposure group).

The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2E+05 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2E+05 cells/mL, unless the mean cell count was less than 3E+05 cells/mL in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (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.

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.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups in the preliminary toxicity test.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test
The dose range of the test item used in the preliminary toxicity test was 9.2 to 2363 µg/mL in all three exposure groups.

There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The onset of test item-induced toxicity was very sharp in all three of the exposure groups. A precipitate of the test item was observed at and above 295.4 µg/mL in the absence of metabolic activation (4-hour and 24-hour exposure). A precipitate of the test item was observed at and above 590.8 µg/mL in the presence of metabolic activation (4-hour exposure). Based on the %RSG values observed, the maximum dose level in the subsequent mutagenicity experiment was limited by test item induced toxicity.

Experiment 1
There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of 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 absence of metabolic activation. Optimum levels of toxicity were not achieved in the presence absence of metabolic activation, despite using a narrow dose interval, due to the very steep toxicity curve of the test item. Whilst optimum levels of toxicity were not achieved a dose level, in the presence of metabolic activation, that exceeded the upper limit of toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. The excessive toxicity observed at and above 25 µg/mL in the absence of metabolic activation, and at and above 60 µg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 50 µg/mL in the in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. 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 dose levels that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. With no evidence of any toxicologically significant increases in mutant frequency in either the absence or presence of metabolic activation in this Experiment, the test item was considered to have been adequately tested. Precipitate of the test item was not observed at any of the dose levels.

Experiment 2
As was seen previously, there was evidence of marked toxicity in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of a reduction in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the RTG and / or %RSG values observed, optimum levels of toxicity were considered to have been achieved in the absence metabolic activation only. Despite using a narrow dose range, optimum levels of toxicity could not be achieved in the presence of metabolic activation due to shift in toxicity. The excessive toxicity observed at and above 40 µg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 56 µg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%. Therefore, this dose level was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a modest effect on the toxicity of the test item.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. 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 1E-06 per viable cell, at any of the dose levels (including the dose level that exceeded the upper limit of acceptable toxicity), in either the absence or presence of metabolic activation. Precipitate of 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'.

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose

(mg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

9.2

106

10

110

18.5

76

88

73

36.9

0

85

8

73.8

0

0

0

147.7

0

0

0

295.4

0

0

0

590.8

0

0

0

1181.5

0

0

0

2363

0

0

0

 

Summary of results

Experiment 1

Treatment

(µg/ml)

4-hours-S-9

Treatment

(µg/ml)

4-hours+S-9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

137.59

 

0

 

100

1.00

128.61

 

2.5

 

94

1.03

115.89

 

2.5

 

98

1.00

117.24

 

5

 

94

0.99

128.48

 

5

 

97

1.04

108.62

 

10

 

86

0.98

94.03

 

10

 

94

0.88

115.31

 

20

 

22

0.27

104.22

 

20

 

80

0.97

108.70

 

25

Ø

1

 

 

 

40

 

37

0.36

117.40

 

30

Ø

0

 

 

 

50

X

8

0.07

149.70

 

35

Ø

0

 

 

 

60

Ø

1

 

 

 

40

Ø

0

 

 

 

70

Ø

0

 

 

 

Linear trend

 

NS

Linear trend

 

NS

EMS

 

 

 

 

 

CP

 

 

 

 

 

400

 

57

0.48

839.87

 

2

 

37

0.19

1036.19

 

 

 

 

 

 

 

 

 

 

 

 

 

Experiment 2

Treatment

(µg/ml)

24-hours-S-9

Treatment

(µg/ml)

4-hours+S-9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

126.54

 

0

 

100

1.00

146.88

 

2

 

116

0.95

145.67

 

2

Ø

92

 

 

 

4

 

108

0.93

130.01

 

4

Ø

93

 

 

 

8

 

93

1.01

111.55

 

8

 

90

0.98

107.82

 

16

 

64

0.83

113.49

 

16

 

94

1.00

142.45

 

24

 

32

0.45

122.83

 

32

 

90

1.01

127.00

 

32

 

7

0.14

143.07

 

40

 

87

0.90

132.42

 

40

Ø

1

 

 

 

48

 

39

0.46

150.35

 

48

Ø

0

 

 

 

56

X

4

0.05

182.95

 

Linear trend

 

NS

Linear trend

 

NS

EMS

 

 

 

 

 

CP

 

 

 

 

 

150

 

53

0.42

1075.60

 

2

 

56

0.35

1389.62

 

Cell and 96-Well Plate Counts: Experiment 1 (-S9) 4-Hour Exposure

Treatment

(µg/ml)

Cell counts $

Viability § after

day 2

2 cells/well

Resistant mutants §

after day 2

2000 cells/well

 

 

0h

  24h

48h

 

 

 

 

 

 

 

 

0

A

B

8.81

8.83

7.65

7.99

7.24

6.34

72

74

73

75

85

76

77

85

17

18

25

18

18

14

19

25

2.5

A

B

8.28

8.36

8.65

8.27

6.03

6.48

80

76

87

77

 

 

 

 

21

15

19

17

 

 

 

 

5

A

B

8.62

8.85

8.17

8.32

6.48

5.71

76

82

74

83

 

 

 

 

22

13

26

15

 

 

 

 

10

A

B

8.39

8.20

8.94

8.39

5.34

5.90

83

82

80

78

 

 

 

 

12

18

15

16

 

 

 

 

20

A

B

6.80

4.99

3.69

3.85

4.66

4.82

82

81

87

80

 

 

 

 

22

14

18

17

 

 

 

 

25

A

B

2.32

2.28

1.56(2.32)

1.74(2.28)

1.25(1.56)

1.52(1.74)

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

30

A

B

1.74

1.55

1.18(1.74)

1.05(1.55)

0.81(1.18)

0.73(1.05)

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

35

A

B

1.40

1.66

0.85(1.40)

0.94(1.66)

0.49(0.85)

0.48(0.94)

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

40

A

B

1.19

0.90

0.68(1.19)

0.46(0.90)

0.42(0.68)

0.25(0.46)

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

Positive Control EMS (µg/ml)

400

A

B

7.93

7.88

6.63

6.59

4.27

5.99

70

73

72

70

 

 

 

 

77

59

70

55

 

 

 

 

Statistical Analysis: Experiment 1 (-S9) 4-Hour Exposure

Treatment

(µg/ml)

 

SG

%RSG

%V

RTG

MF§

0

 

13.27

100

81.33

1.00

137.59

2.5

 

13.23

94

89.59

1.03

115.89

5

 

12.56

94

85.83

0.99

128.48

10

 

12.17

86

91.99

0.98

94.03

20

 

4.47

22

98.08

0.27

104.22

25

Ø

0.57

1

 

 

 

30

Ø

0.21

0

 

 

 

35

Ø

0.11

0

 

 

 

40

Ø

0.05

0

 

 

 

Positive Control EMS

Treatment

(µg/ml)

SG

%RSG

%V

RTG

MF§

400

 

8.48

57

67.78

0.48

839.87

 

 

 

 

 

 

 

Test for linear trend

Slope

‑1.664E‑006

Variance

7.174E‑013

b²/Sb

3.861

Conclusions:
The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
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. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In VitroMammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

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 eight dose levels in duplicate, together with vehicle, dimethyl sulfoxide (DMSO), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

 

Experiment 1

Group

Concentration of OxyoctalineFormate (µg/mL) plated for mutant frequency

4-hour without S9

2.5, 5, 10, 20,

4-hour with S9 (2%)

2.5, 5, 10, 20, 40, 50

 

Experiment 2

Group

Concentration of OxyoctalineFormate (µg/mL) plated for mutant frequency

24-hour without S9

2, 4, 8, 16, 24, 32

4-hour with S9 (2%)

8, 16, 32, 40, 48, 56

 

Results…..

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle controls (DMSO) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

 

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

 

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

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:
The study was conducted between 27 May 2014 and 17 September 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Batch: SC00010054
Purity: 96.5%
Physical state/Appearance: Clear colourless liquid
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking 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 mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Test Item
The molecular weight of the test item was given as 236.3 g/mol, therefore, the maximum dose level was 2363 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. The purity of the test item was 96.5% and was accounted for in the test item formulations.

The test item was insoluble in aqueous media at 23.63mg/mL but was miscible in dimethyl sulphoxide (DMSO) at 236.3 mg/mL in solubility checks performed in house. The test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.

The solubility of the test item was investigated in the Harlan Laboratories Ltd Mouse Lymphoma assay, Study number 41400641.

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Vehicle / solvent:
The test item was insoluble in aqueous media at 23.63mg/mL but was miscible in dimethyl sulphoxide (DMSO) at 236.3 mg/mL in solubility checks performed in house.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Without S9: mitomycin C (MMC) in Minimal Essential Medium at 0.4 and 0.2 µg/mL for cultures in Experiment 1 and 2 respectively. With S9: cyclophosphamide (CP) in dimethyl sulphoxide at 5 µg/mL in both experiments.
Details on test system and experimental conditions:
Cell culture
Cells (whole blood cultures) 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 heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Test Procedure
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:

9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized 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 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL 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 0.1 mL of the appropriate vehicle control, test item 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 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item 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.

The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

Preliminary Toxicity Test
Three exposure groups were used:

i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

The dose range of test item used was 9.2 to 2363 µg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item 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 item toxicity and for selection of the dose levels for the main test.

Experiment 1
Two exposure groups were used for Experiment 1:

i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 4.63 to 74 µg/mL.

ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 9.25 to 148 µg/mL.

Experiment 2
Two exposure groups were used for Experiment 2:

i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 4.63 to 74 µg/mL.

ii) 4-hour exposure to the test item with S9-mix (1%) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 1.16 to 74 µg/mL.

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 prior to slide preparation.

Preparation of Metaphase Spreads
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. Each slide was permanently labeled with the appropriate identification data.
Staining
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 item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator. Supplementary slides were coded manually.

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 at least 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) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. If a weak or equivocal polyploidy effect was observed then an additional independent evaluation of the frequency of polyploidy cells was considered. In addition, the incidence of cells with endoreduplicated chromosomes was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.
Evaluation criteria:
The following criteria were used to determine a valid assay:

Negative Control
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.

Positive Control
All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.
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. (Richardson et al. 1989).
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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 9.2 to 2363 µg/mL. The maximum dose was the maximum recommended dose level, the 10 mM concentration.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 147.7 µg/mL, in both of the 4(20)-hour exposure groups, and at and above 295.4 µg/mL in the 24-hour continuous exposure group.

Haemolysis was observed following exposure to the test item at and above 36.9 µg/mL in all three of the exposure groups. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 73.8 µg/mL and 36.9 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9) respectively. The maximum dose with metaphases present in the 24-hour continuous exposure was 73.8 µg/mL. The test item induced evidence of toxicity in three exposure groups.
The selection of the maximum dose level was based on toxicity in both experiments.

The qualitative assessment of the slides determined that the toxicity was marginally greater to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 37 µg/mL in the absence of metabolic activation, and 74 µg/mL in the presence of metabolic activation (S9).

No precipitate was observed at the end of exposure in either of the exposure groups. Haemolysis was observed following exposure to the test item at and above 18.5 µg/mL in the absence of S9 and at and above 37 µg/mL in the presence of S9.
The mitotic index data are given. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 49% mitotic inhibition was achieved at 37 µg/mL in the absence of S9. In the presence of S9 dose-related inhibition of mitotic index was observed with 58% being achieved at 74 µg/mL.

The maximum dose level selected for metaphase analysis was based on toxicity for both exposure groups, and was 37 and 74 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. 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 aberrations in either the absence or presence of metabolic activation.

The test item 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 up to 55.5 µg/mL in the absence and presence of S9.

No precipitate was observed at the end of exposure in either of the exposure groups. Haemolysis was observed following exposure to the test item at and above 37 µg/mL in both exposure groups.

The mitotic index data are given. They confirm the qualitative observations in that dose-related inhibition of mitotic index was observed, and that 70% mitotic inhibition was achieved at 55.5 µg/mL in the absence of S9. In the presence of S9 dose-related inhibition of mitotic index was observed with 74% inhibition being achieved at 55.5 µg/mL.

The maximum dose level selected for metaphase analysis was based on toxicity. Whilst the level of toxicity observed at 55.5 µg/mL in both exposure groups markedly exceeded the optimum 50% level with no marked evidence of toxicity at the next dose down this dose level was selected for both exposure groups.

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 in either the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test item are given in the table below:

 

Group

Final concentration ofOxyoctalineFormate(µg/mL)

4(20)-hour without S9

0*, 4.63, 9.25*, 18.5*, 27.8*, 37*, 55.5, 74, MMC0.4*

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

0*, 9.25*, 18.5*, 37*, 74*, 111, 148, CP 5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test item are given in the table below:

 

Group

Final concentration ofOxyoctalineFormate(µg/mL)

24-hour without S9

0*, 4.63, 9.25*, 18.5*, 37*, 55.5*, 74, MMC0.2*

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

0*, 1.16, 2.31, 4.63, 9.25*, 18.5*, 37*, 55.5*, 74, CP5*

*  = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Mitotic Index - Preliminary Toxicity Test

4-Hour Treatment, 20-Hour Recovery -S9

4-Hour Treatment, 20-Hour Recovery +S9

24-Hour Treatment -S9

Dose Level

(µg/mL)

4(20)-Hour Without S9

4(20)-Hour With S9

24-Hour Without S9

Mitotic Index

% of Control

Mitotic Index

% of Control

Mitotic Index

% of Control

0

6.55

100

2.40

100

4.70

100

9.2

7.15

109

-

-

-

-

18.5

4.90

75

3.30

138

33.95

84

36.9

3.60 H

55

3.20 H

133

2.40 H

51

73.8

NM H

0

1.55 H

65

1.15 H

24

147.7

NM P H

0

NM P H

0

NM H

0

295.4

NM P H

0

NM P H

0

NM P H

0

590.8

NM P H

0

NM P H

0

NM P H

0

1181.5

NM P H

0

NM P H

0

NM P H

0

2363

NM P H

0

NM P H

0

NM P H

0

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

P = Precipitate observed at end of exposure period in blood-free cultures

H = Haemolysis observed at the end of exposure period.

Mitotic Index - Experiment 1

Dose Level (mg/mL)

4(20)-Hour Without S9

4(20)-Hour With S9

A

B

Mean

% of Control

A

B

Mean

% of Control

0

2.60

2.05

2.33

100

1.30

3.20

2.25

100

4.63

-

-

-

-

NA

NA

NA

NA

9.25

0.80

1.95

1.38

59

2.85

3.15

3.00

133

18.5

1.55 H

4.30 H

2.93

126

4.25

5.90

5.08

226

27.8

5.05 H

2.25 H

3.65

157

NA

NA

NA

NA

37

1.20 H

1.15 H

1.18

51

2.35 H

4.80 H

3.58

159

55.5

NM H

NM H

NM

0

NA

NA

NA

NA

74

NM H

NM H

NM

0

1.00 H

0.90 H

0.95

42

111

NA

NA

NA

NA

NM H

NM H

NM

0

148

NA

NA

NA

NA

NM H

NM H

NM

0

MMC 0.4

2.25

1.50

1.88

81

NA

NA

NA

NA

CP 5

NA

NA

NA

NA

0.65

0.35

0.50

22

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

H = Haemolysisobserved at the end of exposure period.

Mitotic Index - Experiment 2

Dose Level

(µg/mL)

24-Hour Without S9

4(20)-Hour With S9

A

B

Mean

% of Control

A

B

Mean

% of Control

0

1.95

3.25

2.60

100

6.70

7.20

6.95

100

1.16

NA

NA

NA

NA

-

-

-

-

2.31

NA

NA

NA

NA

-

-

-

-

4.63

-

-

-

-

-

-

-

-

9.25

3.60

2.40

3.00

115

7.05

8.15

7.60

109

18.5

1.80

2.65

2.23

86

6.85

7.15

7.00

101

37

2.60 H

3.15 H

2.88

111

6.00 H

6.35 H

6.18

89

55.5

0.65 H

0.90 H

0.78

30

1.20 H

2.40 H

1.80

26

74

NM H

NM H

NM

0

NM

NM

NM

0

MMC 0.2

1.00

1.20

1.10

42

NA

NA

NA

NA

CP 5

NA

NA

NA

NA

2.15

1.65

1.90

27

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

H = Haemolysisobserved at the end of exposure period.

Mean Frequency of Polyploid Cells (%)

Experiment 1

Dose Level

(µg/mL)

Exposure Group

4(20)-Hour Without S9

4(20)-Hour With S9

0

1.0

0.0

9.25

0.0

0.0

18.5

0.0

0.0

27.8

0.0

NA

37

0.0

0.0

74

NA

0.0

MMC 0.4

0.0

NA

CP 5

NA

0.0

 

Experiment 2

Dose Level

(µg/mL)

Exposure Group

24-Hour Without S9

4(20)-Hour With S9

0

0.0

0.0

9.25

0.0

0.0

18.5

0.0

0.0

37

0.0

0.0

55.5

0.0

0.0

MMC 0.2

0.0

NA

CP 5

NA

0.0

MMC    Mitomycin C

CP         Cyclophosphamide

NA    Not applicable

Conclusions:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item, Oxyoctaline Formate 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 identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991). 

 

Methods…….

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

 

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 Oxyoctaline Formate (µg/mL)

4(20)-hour without S9

4.63, 9.25, 18.5, 27.8, 37, 55.5, 74

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

9.25, 18.5, 37, 74, 111, 148

24-hour without S9

4.63, 9.25, 18.5, 37, 55.5, 74

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

1.16, 2.31, 4.63, 9.25, 18.5, 37, 55.5, 74

 

Results…….

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

 

All 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 toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded approximately 50% mitotic inhibition.

 

Conclusion

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

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:
The study was conducted between 30 October 2003 and 21 November 2003.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guidline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch No.: 9000502919
Purity: 99.5%
Colour: Colourless to pale yellow
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: See below.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9 is used as the metabolic activation system.
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since relevant toxic effects were only observed at high concentrations and 5000 µg/plate were chosen as maximal concentration of the main experiments. Strong toxic effects were observed in the second experiment especially in strain TA 1537 with metabolic activation and strain TA 102 without metabolic activation. Therefore, the second experiment was repeated (experiment 2a) with these test groups using an adjusted concentration range. The concentration range included two logarithmic decades. The following concentrations were tested:
Experiment 1: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment 2: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment 2a: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
On the day of the experiment, the test item OXYOCTALlNE FORMATE was dissolved in ethanol (purity> 99 %, MERCK, 0-64293 Oarmstadt). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed.
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed.
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene
Details on test system and experimental conditions:
Pre-cultures
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 µL tetracycline (2 µg/mL) was added to strain TA 102. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCI.
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 C.
Selective Agar
The plates with the minimal agar were obtained from E. Merck.

Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar
6.0 g NaCI
10.5 mg L-Histidine x HCI x H20
12.2 mg Biotin
Sterilisation was performed at 121° C in an autoclave.

Pre-experiment for toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all of the strains. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment 1 below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment 1, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Experimental performance
For each strain and dose level, including the controls three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL Overlay agar
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark
Evaluation criteria:
Acceptability of the Assay
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not required.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Relevant toxic effects, evident as a dose dependent reduction of the number of revertants below half of the colony count of the solvent control, occurred in strain TA 98 and TA 102 of the first experiment at 5000 µg/plate with metabolic activation.
In experiments 2 and 2a such toxic effects also occurred.

Impaired bacterial background growth as the second parameter of toxicity was generally observed at the same concentrations.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. The historical positive control range was slightly exceeded in strain TA 1535 without metabolic activation and in strain TA 1537 with metabolic activation. This effect was considered to emphasise the sensitivity of the strains rather than compromising the study.
The solvent control in strain TA 100 with metabolic activation slightly exceeded the historical range of solvent controls (220 and 212 versus 208 colonies). This minor deviation was considered as irrelevant fluctuation since the corresponding negative control remained within the historical range of negative controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiment 1: Plate incorporation test - TA 1535

Concentration

(µg/plate)

Plate

Revertants/plate

1

2

3

Mean

s.d.

Factor*

Without S9 mix

Negative control

14

18

22

18

4.0

 

Solvent control

15

24

20

20

4.5

1.0

Positive control**

832

647

970

816

162.1

41.5

33

17

17

13

16

2.3

0.8

100

13

17

16

15

2.1

0.8

333

21

23

21

22

1.2

1.1

1000

21

17

15

18

3.1

0.9

2500

25

26

25

25

0.6

1.3

5000

25

25

22

24

1.7

1.2

With S9 mix

Negative control

16

14

13

14

1.5

 

Solvent control

21

17

133

17

4.0

1.0

Positive control***

387

394

460

414

40.3

24.3

33

17

18

19

18

1.0

1.1

100

15

11

17

14

3.1

0.8

333

11

16

15

14

2.6

0.8

1000

15

17

21

18

3.1

1.0

2500

14

11

11

12

1.7

0.7

5000

10

12

14

12

2.0

0.7

*= enhancement factor = (Σrevertants/concentration test item) / (Σrevertants/solvent control)

** = sodium azide 10µg/plate

*** = 2-aminoanthracene 2.5µg/plate

Experiment 1: Plate incorporation test - TA 1537

Concentration

(µg/plate)

Plate

Revertants/plate

1

2

3

Mean

s.d.

Factor*

Without S9 mix

Negative control

6

6

6

6

0.0

 

Solvent control

6

8

8

7

1.2

1.0

Positive control**

64

77

59

67

9.3

9.1

33

5

7

10

7

2.5

1.0

100

7

8

7

7

0.6

1.0

333

8

10

4

7

3.1

1.0

1000

8

8

5

7

1.7

1.0

2500

7

8

6

7

1.0

1.0

5000

4

3

5

4

1.0

0.5

With S9 mix

Negative control

6

6

13

8

4.0

 

Solvent control

4

11

5

7

3.8

1.0

Positive control***

382

400

422

401

20.0

60.2

33

9

6

8

8

1.5

1.1

100

8

10

10

9

1.2

1.4

333

5

5

10

7

2.9

1.0

1000

10

8

12

10

2.0

1.5

2500

5

7

9

7

2.0

1.1

5000

7

4

4

5

1.7

0.8

*= enhancement factor = (Σrevertants/concentration test item) / (Σrevertants/solvent control)

** = 4 -nitro-o-phenylene-diamine 50 µg/plate

*** = 2-aminoanthracene 2.5 µg/plate

Experiment 1: Plate incorporation test - TA 98

Concentration

(µg/plate)

Plate

Revertants/plate

1

2

3

Mean

s.d.

Factor*

Without S9 mix

Negative control

31

27

31

30

2.3

 

Solvent control

31

34

36

34

2.5

0.1

Positive control**

286

311

285

294

14.7

8.7

33

28

21

24

24

3.5

0.7

100

24

27

28

26

2.1

0.8

333

26

22

26

25

2.3

0.7

1000

25

23

28

25

2.5

0.8

2500

22

29

27

26

3.6

0.8

5000

26

28

25

26

1.5

0.8

With S9 mix

Negative control

29

35

20

28

7.5

 

Solvent control

39

36

27

34

6.2

1.0

Positive control***

857

670

577

701

142.6

20.6

33

40

30

33

34

5.1

1.0

100

23

26

22

24

2.1

0.7

333

22

31

23

25

4.9

0.7

1000

31

22

20

24

5.9

0.7

2500

27

25

29

27

2.0

0.8

5000

12

17

10

13

3.6

0.4

*= enhancement factor = (Σrevertants/concentration test item) / (Σrevertants/solvent control)

** = 4 -nitro-o-phenylene-diamine 50µg/plate

*** = 2-aminoanthracene 2.5µg/plate

Experiment 1: Plate incorporation test - TA 100

Concentration

(µg/plate)

Plate

Revertants/plate

1

2

3

Mean

s.d.

Factor*

Without S9 mix

Negative control

185

188

179

184

4.6

 

Solvent control

211

186

179

192

16.8

1.0

Positive control**

609

625

588

607

18.6

3.2

33

210

189

208

202

11.6

1.1

100

206

209

203

206

300

1.1

333

152

197

179

176

22.6

0.9

1000

178

177

208

188

17.6

1.0

2500

185

189

166

180

12.3

0.9

5000

137

153

143

144

8.1

0.8

With S9 mix

Negative control

174

187

182

181

6.6

 

Solvent control

225

220

214

220

5.5

1.0

Positive control***

1111

1196

1318

1208

104.0

5.5

33

2.3

247

248

233

25.7

1.1

100

247

221

199

222

24.0

1.0

333

152

150

190

164

22.5

0.7

1000

182

184

140

169

24.8

0.8

2500

141

130

121

131

10.0

0.6

5000

128

117

120

122

5.7

0.6

*= enhancement factor = (Σrevertants/concentration test item) / (Σrevertants/solvent control)

** = sodium azide 10µg/plate

*** = 2-aminoanthracene 2.5µg/plate

Experiment 1: Plate incorporation test - TA 102

Concentration

(µg/plate)

Plate

Revertants/plate

1

2

3

Mean

s.d.

Factor*

Without S9 mix

Negative control

198

190

173

187

12.8

 

Solvent control

135

163

160

153

15.4

1.0

Positive control**

1274

1151

1127

1184

78.9

7.8

33

170

150

149

156

11.8

1.0

100

151

150

146

149

2.6

1.0

333

137

149

177

154

20.5

1.0

1000

139

182

157

159

21.6

1.0

2500

138

154

149

147

8.2

1.0

5000

152

111

117

127

22.1

0.8

With S9 mix

Negative control

244

246

267

252

12.7

 

Solvent control

171

163

190

175

13.9

1.0

Positive control***

1115

1404

1333

1284

150.6

7.4

33

194

190

194

193

2.3

1.1

100

179

187

175

180

6.1

1.0

333

128

122

112

121

8.1

0.7

1000

146

111

123

127

17.8

0.7

2500

124

109

134

122

12.6

0.7

5000

75

81

69

75

6.0

0.4

*= enhancement factor = (Σrevertants/concentration test item) / (Σrevertants/solvent control)

** = methyl methane sulfonate 4µL/plate

*** = 2-aminoanthracene 10µg/plate

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Oxyoctaline formate is considered to be non-mutagenic in this Salmonella typhimirium reverse mutation assay.
Executive summary:

The study was performed to investigate the potential of oxyoctaline formate to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II, IIa) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

The assay was performed with and without liver microsomal activation. Each concentration, including controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I: 33, 100, 222, 1000, 2500 and 5000 µg/plate

Experiment II: 33, 100, 222, 1000, 2500 and 5000µg/plate

Experiment IIa: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate

Relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation at high concentrations.

Many of the plates incubated with the test item showed impaired background growth in experiment II and IIa at high concentrations.

No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment with Oxyoctaline formate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

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

Additional information

Additional information from genetic toxicity in vitro:

The test substance was assessed for genetic toxicity in three in vitro studies.

An in vitro gene mutation study in bacteria was performed according to OECD Test Guideline 471 using a plate incorporation test and a pre-incubation test with and without metabolic activation.

Relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation at high concentrations.

Many of the plates incubated with the test item showed impaired background growth in experiment II and IIa at high concentrations.

No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

An in vitro cytogenicity study in mammalian cellswas conducted according to OECD Test Guideline 473. The method was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

The maximum dose levels used in the mutagenicity test were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels in the mutagenicity test. The vehicle controls (DMSO) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

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

 

An in vitro gene mutation study in mammalian cells was conducted according toOECD Test Guideline 476 for the detection of structural chromosomal aberrations in cultured mammalian cells.

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

All 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 toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded approximately 50% mitotic inhibition.

The test substance was non-clastogenic to human lymphocytesin vitro.


Justification for selection of genetic toxicity endpoint
The study was conducted on the test substance in an appropriate model and according to internationally recognised guidelines.

Justification for classification or non-classification

A mutation is a permanent change in the amount or structure of the genetic material in a cell. The term “mutation” applies to both heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known, including specific base pair changes and chromosomal translocations. The term “mutagenic” and “mutagen” are used for agents giving rise to an increased occurrence of mutations in populations of cells or organisms.

The more generic terms “genotoxic” and “genotoxicity” apply to agents or processes which alter the structure, information content or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non-physiological manner temporarily alter its replication. Genotoxicity test results are usually taken as indicators for mutagenic effects.

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.

The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.

Three in vitro studies were performed on the target substance. The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item did not induce any toxicologically significant increases in the mutant frequency and was non-clastogenic to human lymphocytes in vitro.

The test substance is therefore not classified for genotoxicity.