Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic effects - bacterial: Ames study. Negative. OECD 471 and OECD 472; Reliability = 1.

Clastogenic effects - mammalian: Chromosome aberrations in human peripheral blood lymphocytes. Negative. OECD 473; Reliability = 1.

Mutagenic effects - mammalian: Mouse lymphoma assay. Negative. OECD 476; Reliability = 1

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, 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
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2
Remarks:
pKM101
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from phenobarbital/ß-naphthaflavone-induced SD rats
Test concentrations with justification for top dose:
100, 200, 500, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
Dimethylsulphoxide
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulphoxide
Positive controls:
yes
Positive control substance:
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: Acridine mutagen ICR191 (TA1537/without activation), 2-Aminoanthracene (all strains/with activation), Daunomycin HCl (TA98/without activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Top agar (plate incorporation)
- Cell density at seeding: 0.1 mL aliquots of each bacterial strain

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 3 days
- All plates were counted by an automatic colony counter with the discrimination adjusted appropriately to permit the optimal counting of mutant colonies
Evaluation criteria:
A positive response in a valid individual experiment is achieved when one or both of the following criteria are met: A statistically significant dose-related increase in the mean number of revertant colonies is obtained; a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) which is statistically significant, is observed at one or more concentrations.

A negative result in a valid individual experiment is achieved when: There is no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.
Statistics:
An assessment of statistical significance was carried out using a one-tailed Student's t-test. The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom. Values of p <0.01 are treated as significant, with values of 0.01≤ p <0.05 being indicative of a possible effect.
Key result
Species / strain:
other: all species/strains tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Table-1: Test Data for Experimental Phase – 1

Strain

Metabolic activation

Dose levels (µg/plate)

Mean

Standard deviation

Ratio: Test/control

No Revertants/plate

Plate 1

Plate 2

Plate 3

TA1535

+S9

5000

15.3

1.2

1.2*

16

14

16

2500

10.0

7.8

0.8

6

19

5

1000

13.7

6.7

1.1

18

17

6

500

14.0

7.2

1.1

6

20

16

200

10.0

2.6

0.8

13

9

8

100

13.7

3.8

1.1

11

12

18

TA1535

-S9

5000

6.3

1.5

0.8

8

6

5

2500

12.0

5.3

1.4

10

10

8

1000

9.0

7.9

1.1

6

18

3

500

6.0

4.0

0.7

6

2

10

200

11.7

1.5

1.4*

10

12

13

100

8.3

0.6

1.0

8

8

9

TA1537

+S9

5000

3.3

1.5

1.1

5

2

3

2500

2.3

0.6

0.8

2

2

3

1000

3.0

1.0

1.0

4

2

3

500

5.0

1.0

1.7**

6

5

4

200

5.0

2.6

1.7

8

3

4

100

4.3

1.5

1.4

4

6

3

TA1537

-S9

5000

2.3

0.6

0.6

3

2

2

2500

3.0

1.7

0.8

5

2

2

1000

3.3

1.2

0.8

2

4

4

500

3.7

1.5

0.9

4

5

2

200

3.7

2.1

0.9

2

3

6

100

3.3

0.6

0.8

3

3

4

TA98

+S9

5000

11.7

2.1

0.6

14

11

10

2500

20.7

3.1

1.0

20

18

24

1000

17.3

0.6

0.8

17

18

17

500

18.3

2.5

0.9

21

16

18

200

18.3

2.1

0.9

20

16

19

100

20.3

4.5

1.0

20

25

16

TA98

-S9

5000

17.0

1.0

0.9

18

17

16

2500

15.0

4.6

0.8

19

10

16

1000

14.0

3.0

0.7

11

14

17

500

17.0

1.0

0.9

18

16

17

200

18.7

4.6

1.0

16

16

24

100

20.7

3.8

1.1

18

25

19

TA100

+S9

5000

104.0

12.0

1.2*

104

116

92

2500

77.0

5.3

0.9

73

75

83

1000

85.0

5.3

1.0

89

79

87

500

99.3

7.6

1.1*

91

106

101

200

96.5

4.9

1.1

93

100

C

100

90.7

3.5

1.0

94

91

87

TA100

-S9

5000

80.7

1.5

1.1

82

79

81

2500

81.0

2.0

1.1

79

83

81

1000

85.3

8.4

1.1

95

80

81

500

81.7

8.3

1.1

75

91

79

200

89.3

9.3

1.2*

97

79

92

100

83.7

6.5

1.1

90

84

77

WP2P

+S9

5000

45.7

9.5

35

49

49

53

2500

34.7

0.6

35

35

35

34

1000

48.0

5.3

54

46

46

44

500

47.0

3.5

49

49

49

43

200

46.3

3.8

42

49

49

48

100

45.0

4.4

43

42

42

50

WP2P

-S9

5000

37.3

3.2

35

36

36

41

2500

24.3

4.0

28

20

20

25

1000

33.7

6.5

34

40

40

27

500

33.3

7.1

41

32

32

27

200

34.7

4.9

38

37

37

29

100

32.3

2.1

34

30

30

33

WP2P uvrA

+S9

5000

149.7

22.3

164

151

161

124

2500

109.7

8.4

100

115

115

114

1000

147.3

6.4

140

150

150

152

500

136.3

17.2

124

156

156

129

200

150.3

12.0

151

138

138

162

100

143.7

2.3

145

141

141

145

WP2P uvrA

-S9

5000

93.7

12.4

86

108

108

87

2500

76.0

17.3

57

91

91

80

1000

104.3

3.5

108

104

104

101

500

103.3

9.3

111

93

93

106

200

108.7

23.3

119

125

125

82

100

103.3

4.0

104

99

99

107

*: 0.01≤ p < 0.05, ** p <0.01 [One-sided t-Test assumes Test > Control]

C: Contaminated plate

Table 2: Test Data for Experimental Phase 2 (+S9)

Strain

Dose levels (µg/plate)

Mean

Standard deviation

Ratio: Test/control

No Revertants/plate

Plate 1

Plate 2

Plate 3

TA1535

5000

11.0

2.6

0.9

10

14

9

2500

18.0

1.0

1.4*

19

18

17

1000

14.0

4.0

1.1

14

10

18

500

13.7

2.5

1.1

14

11

16

200

8.7

3.5

0.7

9

5

12

100

18.3

1.2

1.5**

19

19

17

TA1537

5000

3.0

1.7

1.0

5

2

2

2500

3.3

1.2

1.1

4

2

4

1000

3.7

1.2

1.2

3

3

5

500

3.7

1.5

1.2

4

2

5

200

4.3

0.6

1.4*

4

5

4

100

2.3

1.5

0.8

4

2

1

TA98

5000

20.3

4.5

0.8

25

20

16

2500

20.7

2.3

0.8

18

22

22

1000

18.3

3.2

0.7

16

22

17

500

21.7

4.5

0.9

22

17

26

200

19.0

2.6

0.8

22

18

17

100

22.0

4.6

0.9

27

21

18

TA100

5000

76.3

16.5

0.9

60

93

76

2500

82.7

4.0

1.0

82

87

79

1000

80.7

12.0

0.9

80

69

93

500

73.3

5.1

0.8

69

72

79

200

85.7

6.0

1.0

85

80

92

100

84.0

12.0

1.0

96

84

72

WP2P

5000

50.7

11.7

1.0

42

64

46

2500

49.3

3.5

0.9

49

53

46

1000

48.0

10.4

0.9

60

41

43

500

53.0

4.0

1.0

57

49

53

200

56.0

5.2

1.1

59

59

50

100

50.0

4.4

0.9

53

45

52

WP2P uvrA

5000

155.7

8.5

1.1

162

159

146

2500

156.0

4.4

1.1

161

154

153

1000

150.0

4.0

1.0

146

150

154

500

157.3

2.1

1.1*

155

159

158

200

160.0

13.7

1.1

163

172

145

100

147.3

14.0

1.0

148

161

133

*: 0.01≤ p < 0.05, ** p <0.01 [One-sided t-Test assumes Test > Control]

Table 3: Test Data for Experimental Phase 2 (-S9)

Strain

Dose levels (µg/plate)

Mean

Standard deviation

Ratio: Test/control

No Revertants/plate

Plate 1

Plate 2

Plate 3

TA1535

5000

14.0

3.5

1.3

12

12

18

2500

11.7

0.6

1.1

11

12

12

1000

13.0

3.0

1.3

10

16

13

500

9.0

1.7

0.9

11

8

8

200

9.3

1.5

0.9

11

8

9

100

9.0

3.6

0.9

13

8

6

TA1537

5000

2.3

0.6

1.0

3

2

2

2500

2.0

1.0

0.9

3

1

2

1000

2.7

2.9

1.2

6

1

1

500

2.7

1.2

1.2

4

2

2

200

2.3

1.2

1.0

3

1

3

100

2.3

0.6

1.0

2

2

3

TA98

5000

14.7

3.2

0.6

17

11

16

2500

18.7

2.5

0.7

16

21

19

1000

24.3

6.0

0.9

30

18

25

500

23.3

4.9

0.9

21

29

20

200

20.7

6.4

0.8

28

17

17

100

24.7

3.5

0.9

25

28

21

TA100

5000

70.3

5.5

0.9

74

73

64

2500

72.3

3.1

0.9

73

75

69

1000

66.3

5.5

0.9

72

61

66

500

65.3

3.8

0.9

61

68

67

200

66.3

3.8

0.9

68

69

62

100

74.3

8.4

1.0

69

84

70

WP2P

5000

34.7

6.0

1.2

34

29

41

2500

27.3

0.6

1.0

27

28

27

1000

27.7

3.2

1.0

30

29

24

500

30.0

3.5

1.1

34

28

28

200

28.0

7.2

1.0

36

26

22

100

28.3

1.2

1.0

29

27

29

WP2P uvrA

5000

133.7

16.3

1.1

148

116

137

2500

111.3

4.5

0.9

107

111

116

1000

121.7

3.1

1.0

119

121

125

500

108.3

4.5

0.9

108

104

113

200

127.3

9.1

1.0

117

134

131

100

127.7

15.0

1.0

140

132

111

Conclusions:
The test substance was negative (with and without activation) in all strains tested
Executive summary:

The test substance was evaluated in a bacterial mutagenicity assay over a range of concentrations using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2P and WP2P uvrA) in the presence and absence of a rat liver - derived metabolic activation system (S9-mix), following protocols complying with OECD Guideline Numbers 471 and 472 and with the United Kingdom Department of Health and Guidelines (DOH).

In two separate experiments, the test substance did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the tester strains used, either in the presence or absence of S9-mix.

The sensitivity of the test system, and the metabolic activity of the S9-mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e., non-mutagenic in S. typhimurium and E. coli strains in both the presence and absence of S9 -mix.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.

MEDIA USED
- Type: RPMI 1640 media containing L-glutamine and HEPES
Heat inactivated horse-serum was used in order to eliminate a factor which degrades TFT
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from male Sprague Dawley rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
Range finding study: 15.63, 31.25, 62.50, 125.0, 250.0, 500.0 µg/mL (both with and without S9)
Mutation assay: 0, 10, 20, 40, 60, 80, 100, 110, 120, 130 µg/mL (without S9). 0, 10, 20, 40, 60, 80, 90, 100, 110 120, 130 µg/mL (with S9)
Cytotoxicity assessment assay: 0, 10, 20, 40, 60, 80, 100, 110 120, 130, 140, 150, 175 µg/mL (with S9)
Vehicle / solvent:
The test substance was soluble in DMSO at a concentration of at least 200 mg/mL. The solubility limit in culture medium was in the range of 64.44 to 128.9 μg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 3 hours after test article addition.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
Growth media: RPMI 1640 media supplied containing L-glutamine and HEPES

Cell cultures: The master stock of L5178Y tk+/- mouse lymphoma cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each assay, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated 37±1%. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Treatment of cell cultures (mutation and cytotoxicity assays): For 3 hour treatments in the absence and/or presence of S9, at least 10e7 cells in a volume of 18.8 mL tissue culture medium (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were used. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S9 mix or 150 mM KCl was added. Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments) and the final treatment culture volume was 20 mL.
After 3 hours’ incubation at 37 ± 1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and then finally resuspended in 50 mL RPMI 10 medium. Cells were transferred to tissue culture flasks for growth throughout the expression period. The solubility of the test article in culture was assessed, by eye, at the beginning and end of treatment.

Expression period: In the mutation assay and cytotoxicity assessment assay, cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed. During the expression period, subculturing was performed as required with the aim of retaining an appropriate number of cells/flask.
In the Range-Finder Assay, at the end of the expression period, toxicity was assessed by measuring suspension growth and hence relative suspension growth (RSG), compared to the concurrent vehicle control values. Cultures were not plated for viability assessment.
In the mutation assay and cytotoxicity assessment assay, from observations on recovery and growth of the cultures during the expression period, the appropriate cultures were selected to be plated for viability and TFT resistance.

Plating for viability: At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10000 cells/mL in readiness for plating for TFT resistance. The plates were incubated at 37 ± 1ºC in a humidified incubator gassed with 5 ± 1% v/v CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for TFT resistance: At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10000 cells/mL. TFT (300 μg/mL) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. Plates were incubated at 37 ± 1°C in a humidified incubator gassed with 5 ± 1% v/v CO2 in air until scoreable (14 days). In the mutation assay, wells containing clones were identified as above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive.
Evaluation criteria:
The test substance was considered to be mutagenic in this assay if all of the following criteria were met:
The MF of any test concentration exceeded the sum of the vehicle control mutant frequency plus GEF (i.e., control MF + 126); the linear trend test was statistically significant; and any observed response was reproducible under the same treatment conditions.
The test article was considered as positive in this assay if all of the above criteria were met. The test article was considered as negative in this assay if none of the above criteria were met. Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the cytotoxicity range-finder assay, six concentrations were tested in the absence and presence of S9 ranging from 15.63 to 500 μg/mL (limited by solubility in culture medium). Upon addition of the test substance to the cultures, precipitate was observed at the highest four concentrations (62.5 μg/mL and above). Following the 3 hour treatment incubation period, precipitate was observed at the highest two concentrations (250 μg/mL and above). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S9 (250 μg/mL) was retained and the higher concentration was discarded. The highest concentrations to provide >10% RSG were 125 μg/mL in the absence of S9 and 62.5 μg/mL in the presence of S9, which gave 15% and 54% RSG, respectively.

In the mutation assay ten concentrations, ranging from 10 to 150 μg/mL in the absence of S9 and ranging from 10 to 130 μg/mL in the presence of S9, were tested for 3 hours. Upon addition of the test substance to the cultures, precipitate was observed at the highest six concentrations in the absence and presence of S9 (80 μg/mL and above). Following the 3 hour treatment incubation period, precipitate was observed at the highest four concentrations in the absence of S9 only (110 to 150 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence of S9 (110 μg/mL) was retained and higher concentrations were discarded. The highest concentrations analysed were 110 μg/mL in the absence of S9 and 130 μg/mL in the presence of S9, which gave 11% and 33% RTG, respectively. As there was no concentration giving 10-20% RTG for the mutation assay in the presence of S9, further experimental work was performed in the cytotoxicity assessment assay to define the cytoxtoxicity range under this treatment condition with twelve concentrations ranging from 10 to 175 μg/mL. At the end of treatment incubation period, precipitate was observed at 140 μg/mL and above. Mean % RTG at 130 μg/mL was 27% (with marked heterogeneity between replicate cultures) and extreme cytotoxicity (1% RTG) was observed at the higher concentration of 140 μg/mL. These results substantiated the sharp drop in cytotoxicity induced by the test article after reaching this critical concentration threshold.

Table 1: Summary of Mutation Assay Data

3 hour treatment –S9

3 hour treatment +S9

Concentration μg/mL

% RTG

Mutant frequency

Concentration μg/mL

% RTG

Mutant frequency

0

100

70.69

0

100

58.82

10

102

63.21

10

118

66.24

20

79

56.11

20

92

67.17

40

73

68.12

40

42

81.74

60

54

55.97

60

66

58.04

80 P

28

67.51

80 P

48

73.66

100 P

25

75.90

90 P

45

69.53

110 P PP

11

105.09

100 P

46

64.22

 

 

 

110 P

41

69.15

 

 

 

120 P

37

54.11

 

 

 

130 P

33

70.59

MMS: 15

14

670.35

B[a]P: 2

50

457.36

MMS: 20

43

717.92

B[a]P: 3

29

764.96

% RTG: Percent Relative Total Growth

Linear trend test on mutant frequency: p-value = 0.1447, not significant (absence of S9)

Linear trend test on mutant frequency: p-value = 0.4055, not significant (presence of S9)

P: Precipitation observed at time of treatment

PP: Precipitation observed at end of treatment incubation period

 

Table 2: Summary of RTG, Cytotoxicity Assessment Assay - 3 hour treatment in the presence of S9

Concentration μg/mL

Mean % RTG value

Individual % RTG value

0

100

-

20

132

146, 119

40

111

104, 118

60

94

80, 110

80

76

73, 78

100

67

58, 77

110 P

16

2, 50

120 P

39

37, 42

130 P

27

12, 51

140 P, PP

1

<1, 1

B[a]P: 2

140

-

B[a]P: 3

102

-

% RTG: Percent Relative Total Growth

P: Precipitation observed at time of treatment

PP: Precipitation observed at end of treatment incubation period

Bold text indicates marked heterogeneity between replicate cultures.

Conclusions:
The test substance did not induce mutations at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of S9 activation
Executive summary:

The test substance was assayed for the ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation method. The study consisted of a cytotoxicity range-finder assay and a mutation assay, each conducted in the absence and/or presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9). The test substance was formulated in analytical grade dimethyl sulphoxide (DMSO).

A 3-hour treatment incubation period was used for the mutation assay, which was performed in the absence and presence of S9. Concentrations tested were based on the results of a cytotoxicity range-finder assay in which six concentrations were assessed in the absence and presence of S9, ranging from 15.63 to 500 μg/mL (limited by solubility in culture medium). The highest concentrations to provide >10% relative suspension growth (RSG) were 125 μg/mL in the absence of S9 and 62.5 μg/mL in the presence of S9, which gave 15% and 54% RSG, respectively.

No marked changes in osmolality or pH were observed in the range-finder assay at the highest concentration analysed (250 μg/mL), compared to the concurrent vehicle controls.

In the mutation assay ten concentrations, ranging from 10 to 150 μg/mL in the absence of S9 and from 10 to 130 μg/mL in the presence of S9, were tested for 3 hours. At the end of the treatment incubation period, precipitate was observed at 110 μg/mL and above in the absence of S9 only. Two days after treatment the highest concentrations analysed to determine viability and TFT resistance were 110 μg/mL in the absence of S9 and 130 μg/mL in the presence of S9, which gave 11% and 33% relative total growth (RTG), respectively.

Vehicle and positive control treatments were included in the Mutation Assay in the absence and presence of S9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals methyl methane sulphonate (without S9) and benzo[a]pyrene (with S9).

When tested up to the limit of cytotoxicity for 3 hours in the absence of S9 and up to toxic concentrations for 3 hours in the presence of S9, the MF values of the concentrations plated were all less than the sum of the mean control MF plus the global evaluation factor (GEF, 126 mutants per 10e6 viable cells) and there were no statistically significant linear trends, indicating a negative result.

As there was no concentration giving 10-20% RTG for the mutation assay in the presence of S9, further experimental work was performed to define the cytoxtoxicity range under this treatment condition by testing twelve concentrations, ranging from 10 to 175 μg/mL. At the end of the treatment incubation period, precipitate was observed at 140 μg/mL and above. Mean % RTG at 130 μg/mL was 27% (with marked heterogeneity between replicate cultures) and extreme cytotoxicity (1% RTG) was observed at the higher concentration of 140 μg/mL. These results substantiated the sharp drop in cytotoxicity induced by the test article after reaching this critical concentration threshold. Therefore, the mutation assay in the presence of S9 was considered to have evaluated the maximum feasible concentration without inducing severe toxicity (less than 10-20%) RTG as per OECD Guideline 490, therefore the study was subsequently concluded.

Under the conditions of this study, the test substance did not induce mutations at the tk locus of L5178Y mouse lymphoma cells when tested up to the limit of cytotoxicity for 3 hours in the absence of a rat liver metabolic activation system (S9) and when tested up to toxic concentrations in the presence of S9. The test substance is negative in this in vitro mammalian cell mutation assay under the experimental conditions described.


Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
GLP compliance:
yes
Type of assay:
other: In vitro mammalian cytogenicity/chromosome aberration test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
Human blood samples were obtained by venipuncture in lithium heparin tubes on the days of culture initiation from healthy, non-smoking donors, Donor 1 being male and Donor 2 being female. Both donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.

At 0 hours, cultures (10 mL) were established by the addition of 0.5 mL of whole blood to RPMI-1640 (Dutch modification) tissue culture medium supplemented with approximately 10% foetal bovine serum (FBS), 1.0 IU/mL heparin, 100 IU/mL penicillin and 100 µg/mL streptomycin. The lymphocytes were stimulated to enter cell division by addition of phytohaemagglutinin (PHA) and the cultures were maintained at approximately 37°C for 48 hours with gentle daily mixing where possible.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from male SD rats induced with phenobarbital/ß-naphthoflavone
Test concentrations with justification for top dose:
Without S9-mix: 0.5, 1.0, 2.5, 5.0, 7.5, 10.0, 15.0, 20.0 µg/mL
With S9-mix: 1, 5, 10, 20, 30, 40, 50, 60 µg/mL
Vehicle / solvent:
Dimethylsulphoxide
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 68 hours
- Expression time (cells in growth medium): 92 hours
- Culture harvesting: Approximately 2 hours prior to harvesting, the cultures were treated with colcemid at a final concentration of 0.4 µg/mL. 68 or 92 hours after culture establishment the cultures were centrifuged, the supernatant was removed and the cells were re-suspended in approximately 10 mL of 0.075 M KCI at room temperature for approximately 10 minutes. The cultures were centrifuged, the supernatant was removed and the remaining cells were fixed in freshly prepared methanol/glacial acetic acid fixative (3:1 v/v) added dropwise and made up to a volume of approximately 10 mL. The fixative was removed following centrifugation and replaced with freshly prepared fixative. This fixation process was repeated at least twice prior to slide preparation on clean, moist labelled microscope slides. The slides were air dried, stained in filtered Giemsa stain (10% Gurr's R66 in buffered [pH 6.8] double deionized water) for 7 minutes, rinsed in water, air-dried and mounted with coverslips in DPX.

NUMBER OF REPLICATIONS: 2

SLIDE ANALYSIS:
Slides were examined to determine that they were of suitable quality and, where appropriate, the mitotic index was determined by examining 1000 lymphocytes per culture and calculating the percentage of cells in metaphase.

For each donor, both in the presence and absence of S9-mix, duplicate cultures treated with the test substance at three concentrations were selected for chromosomal aberration analysis at the 68 hour sampling time along with the appropriate solvent and positive control cultures. In each case the highest concentration was selected on the basis of a significant reduction in mean mitotic activity and the suitability of the metaphase preparations for chromosomal aberration analysis. In addition, duplicate cultures from the Donor 2 treated with test substance at the highest concentration suitable for chromosomal aberration analysis (but not exceeding that selected at the 68 hour sampling time) in the presence and absence of S9-mix were selected for chromosomal aberration analysis at the 92 hour sampling time along with the appropriate solvent control cultures.

The slides were coded prior to analysis and 100 cells in metaphase, where possible, were analysed from each selected culture for the incidence of structural chromosomal damage.
Evaluation criteria:
The data have been interpreted as follows:
The assay is considered negative if there is no statistically significant increase in the percentage of aberrant cells (at any concentration) above concurrent solvent control values or if there was statistically significant increase in the percentage of aberrant cells above concurrent solvent control values, which falls within the laboratory solvent control range.

The assay is considered positive, if there is an increase in the percentage of aberrant cells, at least at one concentration, which is substantially greater than the laboratory historical solvent control values.
A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described as above may require further evaluation.
Statistics:
The Fisher Exact Probability Test (one-sided) was used to evaluate statistically the percentage of metaphases showing aberrations (excluding cells with only gap-type aberrations). Data from each treatment group, in the presence and absence of S9-mix, was compared with the respective solvent control group value.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was tested over a range of concentrations, both in the presence and absence of S9-mix, in two independent cytogenetic tests. In each case, test substance was found to be biologically active in the test system, causing concentration related reductions in mitotic activity. Significant reductions in mean mitotic activity were observed in the cultures from both Donor 1 (57% +S9-mix; 58% -S9-mix) and Donor 2 (51% +S9-mix; 54% -S9-mix) treated with the highest concentrations of test substance selected for chromosomal aberration analysis at the 68 hour sampling time.

The sensitivity of the test system, and the metabolic activity of the S9-mix employed, were clearly demonstrated by the increases in the frequencies of aberrant cells induced by the positive control agents, mitomycin C and cyclophosphamide.

Table 1: Mean Chromosomal Aberrations and Mitotic Indices in The Absence of Metabolic Activation (S9-Mix) – 68 Hour Sampling Time

Treatment

Mean % Aberrant Cells Excluding Gaps

Aberrations/Cell Excluding Gaps

Mean % Mitotic Index

Donor 1

Solvent control: 5.0 µL/mL

1.50

0.015

12.6

MMC: 0.2 µg/mL

40.00*

0.400

7.4**

Test substance (µg/mL):

 

 

 

5.0

0.50

0.005

5.3

2.5

2.50

0.035

6.4

0.5

1.00

0.010

11.7

Donor 2

Solvent control: 5.0 µL/mL

1.50

0.015

8.1

MMC: 0.2 µg/mL

52.00*

0.720

4.9**

Test substance (µg/mL):

 

 

 

5.0

2.00

0.020

3.7

2.5

2.00

0.020

4.3

0.5

0.00

0.000

8.5

* Statistically significant increase in the percentage of aberrant cells at p <0.01 using Fisher’s Exact Test (one-sided)

** Positive control mitotic index and percentage of aberrant cells are determined from a single culture

Table 2: Mean Chromosomal Aberrations and Mitotic Indices in The Presence of Metabolic Activation (S9-Mix) – 68 Hour Sampling Time

Treatment

Mean % Aberrant Cells Excluding Gaps

Aberrations/Cell Excluding Gaps

Mean % Mitotic Index

Donor 1

Solvent control: 5.0 µL/mL

1.50

0.015

11.0

CP: 50 µg/mL

26.00*

0.320

5.3**

Test substance (µg/mL):

 

 

 

60

1.00

0.010

4.7

30

0.50

0.005

6.3

5

1.00

0.010

10.1

Donor 2

Solvent control: 5.0 µL/mL

0.50

0.005

8.0

CP: 50 µg/mL

36.00*

0.520

3.8**

Test substance (µg/mL):

 

 

 

5.0

1.50

0.015

3.9

30

6.00*

0.060

4.3

5

2.00

0.020

8.8

* Statistically significant increase in the percentage of aberrant cells at p <0.01 using Fisher’s Exact Test (one-sided)

** Positive control mitotic index and percentage of aberrant cells are determined from a single culture

Table-3: Mean Chromosomal Aberrations and Mitotic Indices in The Presence or Absence Of Metabolic Activation (S9-Mix) – 92 Hour Sampling Time

Treatment

Mean % Aberrant Cells Excluding Gaps

Aberrations/Cell Excluding Gaps

Mean % Mitotic Index

Donor 2 (+S9)

Solvent control: 5.0 µL/mL

4.0

0.070

9.0

Test substance: 50 µg/mL

0.5

0.005

7.2

Donor 2 (-S9)

Solvent control: 5.0 µL/mL

3.0

0.055

10.1

Test substance: 5 µg/mL

1.0

0.010

11.2

Conclusions:
The test substance is not clastogenic either in presence or absence of S9-mix
Executive summary:

The test substance was evaluated for its clastogenic potential in an in vitro cytogenetic assay using human lymphocytes from two donors treated in the presence and absence of a rat liver-derived metabolic activation system (S9-mix). Cultures from both donors were harvested at the standard time of 68 hours after culture initiation and additional cultures from Donor 2 were harvested at the later time of 92 hours after culture initiation.

Cultures treated with the test substance at the following concentrations were selected for chromosomal aberration analysis along with the appropriate solvent and positive control cultures.

Donor 1

68 hours

Donor 2

68 hours

Donor 2

92 hours

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

60 µg/mL

5 µg/mL

50 µg/mL

5 µg/mL

50 µg/mL

5 µg/mL

30 µg/mL

2.5 µg/mL

30 µg/mL

2.5 µg/mL

-

-

5 µg/mL

0.5 µg/mL

5 µg/mL

0.5 µg/mL

-

-

Concentration related reductions in mitotic activity were observed in cultures from both donors, thus demonstrating that the test substance is biologically active in this test system.

At the 68 hour sampling time, no statistically or biologically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded in cultures from Donor 1 treated in either the presence or absence of S9-mix or in cultures from Donor 2 treated in the absence of S9-mix.

A small but statistically significant increase in the percentage of aberrant cells was observed in cultures from Donor 2 treated at the intermediate concentration (30 µg/ml) in the presence of S9-mix. This increase was not dose-related or reproducible in another donor and is therefore considered to be of no biological significance.

No statistically or biologically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded at the 92 hour sampling time in cultures from Donor 2 treated in either the presence or absence of S9-mix.

The sensitivity of the test system, and the metabolic activity of the S9-mix employed, were clearly demonstrated by the increases in the percentage of aberrant cells induced by the positive control agents, mitomycin C and cyclophosphamide.

It was concluded that, under the conditions of this assay, the test substance was not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.

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

Genetic toxicity in vivo

Description of key information

Clastogenic effects - mammalian: in vivo mouse micronucleus study; Negative. OECD 474; Reliability = 1

Clastogenic effects - mammalian: in vivo unscheduled DNA synthesis (UDS); Negative. no guideline; Reliability = 2

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mouse bone marrow micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Margate, UK
- Housing: Housed by sex with up to 5 per cage on mobile mouse cage racks
- Diet: ad libitum
- Water: ad libitum
- Age range when supplied: 5-6 weeks for phase-I and 4-7 weeks for phase-II

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Corn oil
- Volume: 20 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: An individual stock suspension of the test substance was prepared in corn oil for each group of animals. The positive control substance was prepared as a solution in physiological saline. All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. The test substance, vehicle and positive control substance were dosed at a volume of 20 mL/kg bw.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Single dose
Dose / conc.:
2 000 mg/kg bw/day
Dose / conc.:
3 200 mg/kg bw/day
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 65 mg/kg
Tissues and cell types examined:
Tissue: Bone marrow
Cell type: Polychromatic erythrocytes
Details of tissue and slide preparation:
Slide preparation: The animals were killed by asphyxiation in halothane Ph. Eur. followed by cervical dislocation 24 and 48 hours after receiving a single oral dose of the test substance. Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide. This procedure was repeated to give four smears of marrow per slide. The slides were allowed to air dry and were stained with polychrome methylene blue and eosin using an automatic staining machine.

Slide analysis: Slides were coded and scored blind. Initially two thousand (2 x 1000) polychromatic erythrocytes were examined for the presence of micronuclei for each animal. Following completion of this analysis an additional 2000 polychromatic erythrocytes per female were analyzed for the presence of micronuclei at the 48 hour sampling time. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of polychromatic to normochromatic erythrocytes in a sample of 1000 erythrocytes.
Evaluation criteria:
The data have been interpreted as follows:
The assay is considered as negative, if there is no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above concurrent vehicle control incidences or if a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle control incidences but which falls within the laboratory historical vehicle control range.

The assay is considered positive, if there is a statistically and biological significant increase in the incidence of micronucleated polychromatic erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences.

An incidence of micronucleated polychromatic erythrocytes which is statically significantly different from the concurrent vehicle control incidences, but less than 3-fold in excess of both historical and concurrent vehicle control incidences may require further evaluation.
Statistics:
Analyses were carried out using the GLM procedure in SAS (1989). Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs: eyes diminished, signs of diarrhoea, subdued nature, hunched posture, ungroomed appearance and distended abdomen
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Mean Incidence of Micronucleated Polychromatic Erythrocytes/1000 Polychromatic Erythrocytes ± Standard Deviation (SD) At Two Sampling Times

Group Mean Animal Data – Males

Group

Treatment

Dose

Mean incidence of MPE/1000 PE ± SD

24 h

48 h

11

Vehicle control

20 mL/kg

2.1 ± 0.8

1.7 ± 0.3

12

Cyclophosphamide

65 mg/kg

26.0 ± 9.4*

 

13

Test substance

2000 mg/kg

2.7 ± 1.2

 

14

Test substance

3200 mg/kg

2.4 ± 1.5

 

15

Test substance

5000 mg/kg

2.3 ± 2.2

2.3 ± 1.4

** Statistically significant increase in micronucleated polychromatic erythrocytes at p <0.01 in the Student’s t-test (one-sided) on transformed data

All means based on 10 observations (2 counts of 1000 PE per animal).

Group Mean Animal Data – Females

Group

Treatment

Dose

Mean incidence of MPE/1000 PE ± SD

24 h

48 h

11

Vehicle control

20 mL/kg

1.6 ± 0.8

0.8 ± 0.8

12

Cyclophosphamide

65 mg/kg

31.0 ± 12.4**

 

13

Test substance

2000 mg/kg

2.3 ± 0.8

 

14

Test substance

3200 mg/kg

2.9 ± 2.2

 

15

Test substance

5000 mg/kg

2.5 ± 1.4

2.7 ± 1.7*

* Statistically significant increase in micronucleated polychromatic erythrocytes at p <0.05 in the Student’s t-test (one-sided) on transformed data

** Statistically significant increase in micronucleated polychromatic erythrocytes at p <0.01 in the Student’s t-test (one-sided) on transformed data

All means based on 10 observations (2 counts of 1000 PE per animal).

Table 2: Mean Percentage of Polychromatic Erythrocytes ± Standard Deviation (SD) At Two Sampling Times

Group Mean Animal Data – Males

Group

Treatment

Dose

Mean % of PE ± SD

24 h

48 h

11

Vehicle control

20 mL/kg

42.6 ± 9.3

51.2 ± 8.5

12

Cyclophosphamide

65 mg/kg

34.9 ± 7.8

 

13

Test substance

2000 mg/kg

35.5 ± 6.8

 

14

Test substance

3200 mg/kg

36.7 ± 4.5

 

15

Test substance

5000 mg/kg

41.7 ± 7.9

38.7 ± 7.6*

* Statistically significant decrease in the percentage of polychromatic erythrocytes at p <0.05 in the Student’s t-test (one-sided) on transformed data

All mean based on 5 observations (1 count of 1000 erythrocytes per animal).

Group Mean Animal Data – Females

Group

Treatment

Dose

Mean % of PE ± SD

24 h

48 h

11

Vehicle control

20 mL/kg

39.8 ± 8.8

50.4 ± 9.7

12

Cyclophosphamide

65 mg/kg

43.4 ± 10.1

 

13

Test substance

2000 mg/kg

30.5 ± 8.7*

 

14

Test substance

3200 mg/kg

40.5 ± 2.5

 

15

Test substance

5000 mg/kg

36.2 ± 9.4

48.5 ± 7.1

* Statistically significant decrease in the percentage of polychromatic erythrocytes at p <0.05 in the Student’s t-test (one-sided) on transformed data

All mean based on 5 observations (1 count of 1000 erythrocytes per animal).

Conclusions:
The test substance was not clastogenic in the mouse bone marrow micronucleus test.
Executive summary:

The test substance has been evaluated for its ability to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice. A single oral dose was given to groups of 5 male and 5 female mice at dose levels of 2000, 3200 and 5000 mg/kg. The latter dose level used represents the limit dose for the assay. Bone marrow samples were taken 24 and 48 hours after dosing.

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over vehicle control values, were seen at the 24 hour sampling time in either male or female mice dosed with test substance or at the 48 hour sampling time in the male mice dosed with test substance.

Although a small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes, over the vehicle control value, was seen at the 48 hour sampling time in the female mice dosed with test substance at 5000 mg/kg, extended analysis of a further 2000 polychromatic erythrocytes per female showed no such increase thus confirming that the small increase observed in the original counts is of no biological significance.

Comparison of the percentage of polychromatic erythrocytes showed no biologically significant differences between the vehicle control and test substance treated mice at any of the dose levels or either of the sampling times investigated.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Under the conditions of this test, the test substance was not clastogenic in the mouse bone marrow micronucleus test.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance was evaluated using an autoradiographic technique, for its ability to induce unscheduled DNA synthesis in the liver of rats. A single oral dose was given to groups of 5 male rats at 3200 and 5000 mg/kg. Two sampling times, 2 and 16 hours post dose were used and 2 independent experiments were carried out at each time point.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
other: Alpk:APfSD
Details on species / strain selection:
The Alderley Park (Alpk:APfSD) rat, which was used in this study, has been shown to be sensitive to a large number of chemicals known to induce DNA repair in vivo. Only male rats were used as there were no published examples of test substances that produce a UDS response in female rats but not male rats. Where comparisons have been, males have been found to respond more strongly than females in this assay.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Zeneca Rodent Breeding Unit, Alderley Park, Macclesfield, Cheshire
- Assigned to test groups randomly: Yes
- Housing: Mobile rat cage racks
- Diet: ad libitum
- Water: ad libitum
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: An individual stock suspension of the test substance was prepared in corn oil for each group of animals. The positive control substance was prepared as a solution in double deionized water. All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. The test substance, vehicle and positive control substance preparations were dosed at a volume of 20 mL/kg bodyweight.
Duration of treatment / exposure:
2 and 16 hours
Frequency of treatment:
Single dose
Dose / conc.:
3 200 mg/kg bw/day
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control used: Dimethylhydrazine dihydrochloride
- Route of administration: Oral
- Doses / concentrations: 30 mg/kg
Tissues and cell types examined:
Liver and hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A determination of the maximum tolerated dose was not conducted for this assay as previously conducted studies on this test substance had shown that a limit dose level of 5000 mg/kg was appropriate for use in the main UDS experiments.

ISOLATION AND CULTURING OF HEPATOCYTES: All animals designated for perfusion were anaesthetized using halothane Ph Eur one at a time. Each animal was maintained under deep anesthesia to prevent any possible recovery. All animals treated with the test substance were examined internally for signs of coloration or abnormalities to organs/tissues.

A 'V' shaped incision was made through both skin and muscle from the center lower abdomen up through the rib cage. The inferior vena cava was ligatured superior to the kidney. The hepatic portal vein and superior vena cava were cannulated with appropriate gauge catheters. The hepatocytes were isolated by a two stage collagenase perfusion technique. A buffer solution was used to flush the liver free of blood and to remove calcium from the desmosomes. A second buffer solution to which calcium chloride and collagenase had been added was used to cause disaggregation of the liver tissue. The liver was then removed, cut open and finely chopped prior to hepatocyte preparation by low speed centrifugation and resuspension in Williams E complete medium. The viability of the hepatocytes was determined using trypan blue.

The hepatocyte suspensions were diluted with Williams E complete medium to give a final cell count of 1.5 x 10e5 cells/mL and transferred onto coverslips placed etched side up in six-well plates. The cultures were placed in a humidified 37°C incubator with a 95% air:5% CO2 (v/v) atmosphere, for at least 90 minutes to enable cell attachment.

The culture medium was aspirated using aseptic technique and the hepatocytes washed with Williams E incomplete medium. Williams E incomplete medium containing 3H-thymidine was added to each well and the dishes were incubated for approximately 4 hours in a 37°C incubator (humidified, 37°C, 95% air:5% CO2 v/v atmosphere). Cultures were then washed three times with Williams E incomplete medium plus thymidine. This 'cold chase' procedure removed excess radiolabel from the cultures. The cultures were then incubated overnight (at least 12 hours) with the same culture medium.

The cultures were then washed once with Williams E incomplete medium or cold phase medium prior to being fixed at least three times with freshly prepared 1:3 glacial acetic acid:absolute alcohol (v/v) followed by four washes with double deionised water. The overslips were mounted, cell side up, on labelled microscope slides.

STAINING: After the exposure period, slides were developed using KODAK D19 developer and Ilford HYPAM fixer. The cells were stained using Meyers Haemalum and eosin Y phloxine.
Evaluation criteria:
Negative: Mean net nuclear grain counts for all test substance treated animals of less than zero.

An occurrence of a mean net nuclear grain count of zero or above in a test substance treated animal is, therefore, considered to be indicative of a UDS response in that animal. Reproducibility of such a response in animals treated concurrently and in an independent experiment is necessary before concluding that the test substance is positive.

Where an individual animal has given rise to a mean net nuclear grain count of at least zero, but this is not fully reproducible, this may require further investigation.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Treatment (mg/kg)

No. of animals

Mean N ± SD

Mean C ± SD

Mean (N-C) ± SD

Mean % Cells in Repair #

2 Hours

Corn oil: 20 mL/kg

2

4.7 ± 2.2

7.6 ± 2.0

-2.9 ± 0.2

1

Test substance: 3200

5

4.7 ± 0.4

7.4 ± 1.1

-2.7 ± 0.8

0

Test substance: 5000

5

5.8 ± 1.6

9.1 ± 2.1

-3.2 ± 1.0

1

DMH.2HCl: 30

2

18.2 ± 0.0

8.1 ± 1.0

10.1 ± 1.1

68

16 Hours

Corn oil: 20 mL/kg

2

6.2 ± 1.1

8.9 ± 1.4

-2.7 ± 0.2

1

Test substance: 3200

5

5.4 ± 0.5

8.4 ± 0.9

-2.9 ± 0.8

1

Test substance: 5000

5

5.3 ± 1.1

8.2 ± 1.1

-2.9 ± 0.6

1

DMH.2HCl: 30

2

22.4 ± 0.8

10.4 ± 1.8

12.0 ± 1.0

81

N ± SD = Mean nuclear grain count ± standard deviation

C ± SD = Mean cytoplasmic grain count ± standard deviation

(N-C) ± SD = Mean net nuclear grain count ± standard deviation

# A cell in repair is one with a net nuclear grain count of ≥5

Conclusions:
The test substance did not induce DNA repair (as measured by UDS) in the rat liver in vivo
Executive summary:

The test substance has been evaluated, using an autoradiographic technique, for its ability to induce unscheduled DNA synthesis (UDS) in the liver of rats. A single oral dose was given to groups of 5 male rats at dose levels of 3200 and 5000 mg/kg. The latter dose level represents the limit dose level of the assay. Two sampling times, 2 hours and 16 hours post-dose were used and 2 independent experiments were carried out at each time point.

The values recorded for the mean net nuclear grain counts and the percentage of cells in repair clearly show that the test substance did not induce DNA repair, as measured by UDS, at either dose level or time point investigated. The test system positive control, dimethylhydrazine dihydrochloride, induced marked increases in UDS, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known genotoxin.

Under the conditions of this test, the test substance did not induce DNA repair (as measured by UDS) in the rat liver in vivo.

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

Additional information

A bacterial reverse mutation assay was conducted using 4 strains of S. typhimurium and 2 strains of E. coli. The initial assay involved the standard plate incorporation protocol using 6 test concentrations over a dose range of 100 to 5000mg per plate. The experiment was conducted both in the presence and absence of an auxiliary metabolic activation system (rat liver S-9 mix). The test substance was subsequently re-tested in all six strains over the same dose range. Under the conditions of this assay, the test substance gave a negative/non-mutagenic response in S. typhimurium strains and E. coli strains in both the presence and absence of a metabolic activation system.

 

An in vitro mammalian cell assay was conducted to evaluate the test substance for its ability to induce forward mutations in mouse lymphoma cells. Two series of exponentially growing suspension cultures of cells were treated in duplicate with solvent control, positive controls, and a range of concentrations of test substance for 4 hours in the presence and absence of an auxiliary metabolic activation system (rat liver S-9 mix).  Under the conditions of this study, the test substance did not induce mutations at the tk locus of L5178Y mouse lymphoma cells when tested up to the limit of cytotoxicity for 3 hours in the absence of a rat liver metabolic activation system (S9) and when tested up to toxic concentrations in the presence of S9. The test substance is negative in this in vitro mammalian cell mutation assay.

 

The test substance was evaluated for clastogenic potential in an in vitro cytogenetic assay using human lymphocytes from 2 donors treated in the presence and absence of a rat liver-derived metabolic activation system. Cultures from both donors were treated 48 hours after culture initiation. Treatments without activation were for 20 hours and those with metabolic activation were for 3 hours followed by a 17-hour post-treatment incubation (harvested 68 hours after culture initiation). Additional cultures from the second donor were incubated for a further 24 hours following a culture medium change and harvested 96 hours after culture initiation. Under the conditions of this assay, the test substance was not clastogenic to cultures of human lymphocytes treated in vitro in either the presence or absence of metabolic activation.

 

An in vivo cytogenetics study was conducted to evaluate whether or not the test substance is capable of inducing clastogenic damage in the mouse bone marrow micronucleus test. The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen. The test substance was tested up to a limit dose of 5000 mg/kg body weight in both male and female mice. No biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over vehicle control values, were seen in either the 24-hour or 48-hour sampling time. Under the conditions of the test, the test substance is not clastogenic in the mouse bone marrow micronucleus test.

 

An in vivo rat liver unscheduled DNA synthesis assay was conducted to evaluate whether or not the test substance was capable of inducing DNA damage, and consequently DNA repair, in the rat liver in vivo. The test substance was tested up to a limit dose of 5000 mg/kg body weight. No significant adverse reactions to treatment were observed for rats dosed with the test substance. The values recorded for the mean net nuclear grain count and the percentage of cells in repair clearly show that the test substance did not induce DNA repair, as measured by UDS, at either sampling time at 2 or 16 hours. The test system positive control, dimethylhydrazine dihydrochloride, induced marked increases in UDS, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known genotoxin. Under the conditions of the test, the test substance did not induce DNA repair, as measured by unscheduled DNA synthesis, in the rat liver in vivo.

Justification for classification or non-classification

The test substance was negative for mutagenicity and clastogenicityin vitro in bacterial and mammalian cells, respectively. Additionally, the test substance was negative when evaluated in vivo in laboratory animals. Based on an assessment of the robust genetic toxicity data for this substance, the substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.