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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Apr - 17 May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
certified by Hessisches Ministerium für Umwelt, Energie, Jugend, Familie und Gesundheit, 1998
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium)
trp operon (WP2 uvrA)
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
sodium azide (+S9: 10 µg/plate in TA 1535, TA 100), 4-nitro-o-phenylene-diamine (+S9: 10 µg/plate in TA 98, 50 µg/plate in TA 1537), methylmethanesulfonate (+S9: 5 µL/plate in WP2 uvrA), 2-aminoanthracene (-S9: 2.5 µg/plate and 10 µg/
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1) and pre-incubation test (Experiment 2)

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: background growth and reduction in the number of revertants

Evaluation criteria:
A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
A test item producing neither a dose related increase in the number of revertants, nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test item is considered mutagenic if in the strains Ta 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No data.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 100 and 1000 - 5000 µg/plate in TA 100 -S9; 1000 - 5000 µg/plate in TA 1535, TA 1537, TA 98, and TA 100 with S9; 2500 - 5000 µg/plate in WP2 uvrA with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
Toxic effects, evident as a reduction in number of revertants, were observed in all strains at concentrations of 1000 µg/plate or above. Additionally toxic effects were seen in strain TA 100 at 100 µg/plate without S9 mix.

Table 1: Ames Test Results - Experiment 1 (Plate incorporation Test)

With or without S9-Mix

Test substance concentration

(µg/plate)

Mean number of revertant colonies per plate

(average of triplicate)

Base-pair substitution type

Frameshift type

Base-pair substitution and other

TA 1535

TA1537

TA 100

TA98

WP2 uvrA

-

Negative control

11

9

164

31

52

-

Vehicle control

14

10

136

30

51

-

33

11

11

161

35

52

-

100

14

13

153

35

58

-

333

17

12

156

40

53

-

1000

18

11

159

35

62

-

2500

15

12

144

40

58

-

5000

14

16

144

36

58

Positive

controls

- S9

Name

SA

4NP

SA

4NP

MMS

Concentrations

(µg/plate)

10

50

10

10

5 µl

Number of colonies/plate

1549

58

625

159

856

 

TA 1535

TA 1537

TA 100

TA98

WP2 uvrA

+

Negative control

17

20

185

40

50

+

Vehicle control

16

14

184

49

52

+

33

21

22

173

40

56

+

100

23

20

145

41

63

+

333

18

17

151

45

60

+

1000

22

22

135

44

52

+

2500

18

26

148

40

60

+

5000

11

13

114

27

40

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(µg/plate)

2.5

2.5

2.5

2.5

10

Number of colonies/plate

263

130

912

477

226

Table 2: Ames Test Results - Experiment 2 (Pre-Incubation Test)

With or without S9-Mix

Test substance concentration

(µg/plate)

Mean number of revertant colonies per plate

(average of triplicate)

Base-pair substitution type

Frameshift type

Base-pair substitution and other

TA 1535

TA1537

TA 100

TA98

WP2 uvrA

-

Negative control

14

6

147

21

42

-

Vehicle control

9

8

133

27

41

-

33

14

8

88

31

42

-

100

10

6

69

23

45

-

333

8

7

87

20

47

-

1000

8

4

63

25

45

-

2500

7

5

67

31

37

-

5000

7

6

72

28

67

Positive

controls

- S9

Name

SA

4NP

SA

4NP

MMS

Concentrations

(µg/plate)

10

50

10

10

5 µl

Number of colonies/plate

618

53

759

189

414

 

TA 1535

TA 1537

TA 100

TA98

WP2 uvrA

+

Negative control

20

12

168

28

51

+

Vehicle control

23

9

150

36

48

+

33

14

12

131

35

71

+

100

14

5

143

33

68

+

333

16

6

90

26

64

+

1000

5

3

59

10

30

+

2500

8

1

4

6

19

+

5000

6

0

0

0

19

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(µg/plate)

2.5

2.5

2.5

2.5

10

Number of colonies/plate

151

63

681

387

218

SA = Sodium azide

4NP = 4-Nitro-o-phenylene-diamine

MMS = Methyl methane sulfonate

2AA = 2-Aminoanthracene

Conclusions:
During the 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.
Executive summary:

This study was performed to investigate the potential of Y-15099 to induce gene mutations according to the OECD guideline 471 in a plate incorporation test (Experiment 1) and a pre-incubation test (Experiment 2) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in 2 independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 0, 33, 100, 333, 1000, 2500, and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation. Toxic effects, evident as a reduction in number of revertants, were observed in all strains at concentrations of 1000 µg/plate or above. Additionally toxic effects were seen in strain TA 100 at 100 µg/plate without S9 mix.

No substantial increase in revertant colony numbers of any of the 5 tester strains was observed following treatment with Y-15099 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:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May - 3 Aug 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
certified by Hessisches Ministerium für Umwelt, Energie, Jugend, Familie und Gesundheit, 1998
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium; SEROMED; Berlin, Germany)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
-S9:
Experiment 1: 5, 10, 15, 25, 37.5, 50 µg/mL (18 hours preparation interval)
Experiment 2: 0.94, 1.88, 3.75, 7.5, 10, 15 µg/mL (18 hours preparation interval) and 3.75, 7.5, 10, 15 µg/mL (28 hours preparation interval)

+S9:
Experiment 1: 5, 10, 20, 40, 80 ,160 µg/mL (18 hours preparation interval)
Experiment 2: 20, 40, 60, 80, 120, 160 µg/mL (28 hours preparation interval)
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 600 and 1000 µg/mL ethylmethanesulphonate (-S9) and 0.7 and 1.0 µg/mL cyclophosphamide (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Exp. I: 4 hours with and without S9 mix; Exp. II: 18 and 28 hours without S9 mix; 4 hours with S9 mix
- Expression time (recovery): Exp. I: 14 hours; Exp. II: 24 hours with S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells): Exp. I: 18 hours; Exp. II: 18 and 28 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL culture medium)

STAIN (for cytogenetic assays): Giemsa (E. Merck, Darmstadt, Germany)

NUMBER OF REPLICATIONS: two independent experiments with duplicates each

NUMBER OF METAPHASES ANALYZED: 200 per experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduced cell numbers and/or mitotic indices

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Evaluation criteria:
A test item is classified as non-clastogenic if:
The number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0% aberrant cells exclusive gaps) and/or no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
The number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0% aberrant cells exclusive gaps) and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in absence of S9 mix at 28.5 µg/mL cell numbers were significantly reduced (30% of control).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRECIPITATION CONCENTRATION:
Insolubility of the test item, seen as oily globules loating on the surface of the culture medium, were observed after treatment with 57 µg/mL and above in the absence and presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES:
In the pre-test 3650 µg/mL was chosen as highest concentration in accordance with OECD Guideline 473.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects indicated by reduced cell numbers and/or mitotic indices below 50% of control were observed in the absence of S9 mix only. In the presence of S9 mix no relevant toxicity was observed in the pre-test on toxicity.

Table 1: Summary of results of the chromosomal aberration study with Y-15099

Exp.

Preparation interval

Concentration

Polyploid cells in %

Cell numbers in % of control

Mitotic indices in % of control

Incl. gaps

Aberrant cells in % excl. gaps

With exchanges

Exposure period 4 h without S9 mix

1

18 h

Neg. control

Solv. control

Pos. Control

5

10

15

2.3

3.3

1.4

1.3

3.6

3.5

n.t.

100

n.t.

109

82

64

100

100

32

76

81

49

1.5

1.5

20.0

2.0

2.5

1.0

0.5

1.0

20.0

1.5

1.0

0.0

0.0

0.0

5.5

0.0

0.0

0.0

Exposure period 18 h without S9 mix

2

18 h

Neg. control

Solv. control

Pos. Control

3.8

7.5

15

2.3

2.6

3.0

3.2

3.0

3.3

n.t.

100

n.t.

69

119

42

100

100

65

90

106

52

1.5

1.5

20.0

1.0

1.5

1.5

1.0

1.0

19.0

0.0

1.0

0.5

0.0

0.0

5.0

0.0

0.0

0.0

Exposure period 28h without S9 mix

2

28 h

Neg. control

Solv. control

Pos. Control

15

3.0

3.2

3.3

3.9

n.t.

100

n.t.

47

100

100

61

63

1.0

2.0

20.0

1.5

1.0

0.5

20.0

1.0

0.0

0.0

1.0

0.5

Exposure period 4 h with S9 mix

1

18 h

Neg. control

Solv. control

Pos. Control

40

80

100

2.5

1.9

3.0

2.5

2.5

2.7

n.t.

100

n.t.

97

85

58

100

100

145

100

71

109

2.0

2.5

16.0

4.0

3.5

2.0

1.0

1.0

15.0

2.0

2.5

0.5

0.0

1.0

4.5

1.0

1.0

0.0

2

28 h

Neg. control

Solv. control

Pos. Control

40

80

100

3.3

3.8

3.6

3.3

2.8

2.9

n.t.

100

n.t.

75

65

76

100

100

95

101

103

95

4.0

0.5

16.5

1.0

3.0

1.0

2.5

0.5

16.0

1.0

1.0

0.5

0.0

0.0

4.0

0.5

0.0

0.0

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, Y-15099 is considered to be non-clastogenic in this chromosome aberration test neither with nor without S9 mix.
Executive summary:

The test item Y-15099, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were set up.

Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (3650 µg/mL = 10 mM) was chosen with regard to the molecular weight of the test item and the current OECD Guideline 473.

Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of the test item insolubility in the culture medium. The chosen treatment concentrations were:

-S9:

Experiment 1: 5, 10, 15, 25, 37.5, 50 µg/mL (18 hours preparation interval)

Experiment 2: 0.94, 1.88, 3.75, 7.5, 10, 15 µg/mL (18 hours preparation interval) and 3.75, 7.5, 10, 15 µg/mL (28 hours preparation interval)

 

+S9:

Experiment 1: 5, 10, 20, 40, 80 ,160 µg/mL (18 hours preparation interval)

Experiment 2: 20, 40, 60, 80, 120, 160 µg/mL (28 hours preparation interval)

 

Toxic effects indicated by reduced cell numbers and/or mitotic indices below 50% of the control were observed in the absence of S9 mix only. In the presence of S9 mix no relevant toxicity was observed in the pre-test on toxicity. Therefore, the test item was tested up to a concentration exhibiting clear test item insolubility as recommended in the OECD Guideline 473.

In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Jul 2007 - 12 Mar 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
GLP compliance:
yes (incl. QA statement)
Remarks:
certified by Bayerisches Landesamt für Arbeitsschutz, Arbeitsmedizin und Sicherheitstechnik, 2004
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
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:
+S9: 200, 300, 350, 375, 400, 440, 460 and 480 µg/mL
-S9: 14, 18, 20, 22, 24, 26, 28 and 30 µg/mL

Experiment 2:
+S9: 300, 410, 440, 455, 470, 485, 500 and 515 µg/mL
-S9: 2, 14, 22, 26, 30, 33, 36 and 39 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
+S9: benzo(a)pyrene (2.5 µg/mL); -S9: ethylmethanesulphonate (500 µg/mL), methylmethanesulfonate (10 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Exp.I: 4 hours with and without metabolic act.; Exp. II: 4 hours with metabolic act. and 24 hours without metabolic act.
- Expression time (cells in growth medium): 72 hours, when exposure was 4 hours; 48 hours, when exposure was 24 hours.
- Selection time (if incubation with a selection agent): Cultures were seeded in selective medium for a period of 11-14 days.

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 independent experiments (each with four 96-well-plates)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)

After expression, the relative cloining efficiency, relative suspension and total growth were determined.

Evaluation criteria:
There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency,
- biologically relevant response (at least 2-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) for at least one of the dose groups,
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ration (ration of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5) is an indication for potential clastogenic effects and/or chromosomal aberrations.
A test item is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.
Statistics:
No data.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: +S9: at = 375 µg/mL and -S9: at = 24 µg/mL; Experiment 2: +S9: at = 500 µg/mL and -S9: at = 30 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Pre-Test for Toxicity:
The toxicity of the test item was determined in a pre-experiment. Six concentrations [0.1, 0.5, 3, 10, 25 and 50 µg/mL] were tested without metabolic activation and four concentrations were tested with metabolic activation [250, 350, 450 and 550 µg/mL].

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I with S9 mix the relative total growth (RTG) was 19.17% for the highest concentration. Without S9 mix RTG was 13.44% for the highest concentration. In experiment II with S9 mix RTG was 21.91% for the highest concentration and without S9 mix RTG was 12.93% for the highest concentration.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item.

Table 1: Experiment 1 - Mutagenicity Data

 

Cloning Efficiency (CE)

Mutagenicity Data

With metabolic activation (+S9)

Test group

Concentration [µg/mL]

Plate 1

Plate 2

RCE [%]

Mean number of cultures/96 wells

Mutants/106cells

Mutation factor

NC1

 

77

83

101.27

13.50

67.67

 

NC2

84

86

107.59

16.25

68.48

S1

0

78

82

100.00

17.75

91.28

 

S2

82

74

100.00

12.00

63.82

2

200

78

77

98.10

14.75

81.05

1.05

6

300

89

83

108.86

12.00

47.23

0.61

8

350

67

59

79.75

14.75

124.98

1.61

9

375

73

75

93.67

17.00

105.83

1.36

10

400

86

83

106.96

14.50

61.73

0.80

12

440

80

75

98.10

18.00

100.88

1.30

13

460

74

72

92.41

14.50

91.68

1.18

14

480

73

75

93.67

18.00

112.75

1.45

B[a]P

2.5

80

87

105.70

48.25

274.06

3.53

Without metabolic activation (-S9)

NC1

0

87

87

114.85

12.75

48.16

 

NC2

77

73

99.01

12.75

75.01

S1

0

72

73

100.00

14.25

91.34

 

S2

79

79

100.00

12.00

61.71

2

14

87

89

116.17

11.25

40.13

0.52

4

18

90

87

116.83

15.25

54.28

0.71

5

20

71

80

99.67

15.25

89.64

1.17

6

22

75

80

102.31

12.25

66.33

0.87

7

24

84

79

107.59

12.75

60.31

0.79

8

26

74

78

100.33

12.00

68.10

0.89

9

28

83

77

105.61

18.25

94.14

1.23

10

30

77

77

101.65

15.25

85.43

1.12

EMS

500

76

76

100.33

79.00

882.89

11.54

MMS

10

80

74

101.65

58.75

467.53

6.11

Table 2: Experiment 2 - Mutagenicity Data

 

Cloning Efficiency (CE)

Mutagenicity Data

With metabolic activation (+S9)

Test group

Concentration [µg/mL]

Plate 1

Plate 2

RCE [%]

Mean number of cultures/96 wells

Mutants/106cells

Mutation factor

NC1

 

91

84

111.11

16.00

60.17

 

NC2

83

83

105.40

16.00

72.95

S1

0

79

82

100.00

15.75

78.62

 

S2

79

75

100.00

13.25

73.35

2

300

82

83

104.76

15.50

71.81

0.95

6

410

83

83

105.40

14.50

65.52

0.86

8

440

87

87

110.48

16.25

62.68

0.82

9

455

81

88

107.30

12.00

50.34

0.66

10

470

85

90

111.11

14.25

53.02

0.70

12

485

86

91

112.38

13.75

48.51

0.64

13

500

82

82

104.13

13.75

64.23

0.85

14

515

87

85

109.21

12.75

50.40

0.66

B[a]P

2.5

91

85

111.75

48.75

228.22

3.00

Without metabolic activation (-S9)

NC1

0

85

85

99.42

13.50

55.96

 

NC2

86

79

96.49

11.25

50.83

S1

0

89

88

100.00

15.00

53.31

 

S2

84

81

100.00

14.50

66.78

2

2

81

83

95.91

16.25

77.06

1.28

4

14

84

90

101.75

13.50

51.22

0.85

5

22

85

86

100.00

13.25

53.69

0.89

6

26

84

83

97.66

17.25

77.73

1.29

7

30

87

81

98.25

13.00

55.98

0.93

8

33

89

83

100.58

11.25

44.09

0.73

9

36

87

89

102.92

17.50

64.79

1.08

10

39

83

85

9825

13.00

55.98

0.93

EMS

200

59

64

71.93

82.75

1548.07

25.78

MMS

10

65

69

78.36

59.00

637.19

10.61

NC: Negative control

S: Solvent control

RCE: [(mean value positive cultures / mean value positive cultures of corresponding controls) x 100]

B[a]P: Benz(a)pyrene

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Y-15821 is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Executive summary:

The test item Y-15821 was assessed in a GLP study according to OECD 476 for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations was based on data from the pre-experiment. In experiment 1 480 µg/mL (+S9) and 30 µg/mL (-S9) were selected as the highest concentrations. In experiment 2 515 µg/mL (+S9) and 39 µg/mL (-S9) were selected as the highest concentrations. Experiment 2 without metabolic activation was performed as 24 h long time exposure assay.

The test item was investigated at the following concentrations:

Experiment 1:

+S9: 200, 300, 350, 375, 400, 440, 460 and 480 µg/mL

-S9: 14, 18, 20, 22, 24, 26, 28 and 30 µg/mL

Experiment 2:

+S9: 300, 410, 440, 455, 470, 485, 500 and 515 µg/mL

-S9: 2, 14, 22, 26, 30, 33, 36 and 39 µg/mL.

Growth inhibition was observed in experiment 1 and 2 with and without metabolic activation.

In experiment 1 with metabolic activation the relative total growth (RTG) was 19.17% for the highest concentration (480 µg/mL) evaluated. The highest biologically relevant concentration evaluated –S9 mix was 30 µg/mL with a RTG of 13.44%. In experiment 2 +S9 mix the RTG was 21.91% for the highest concentration (515 µg/mL) evaluated. The highest biologically relevant concentration evaluated – S9 mix was 39 µg/mL with a RTG of 12.93%.

In Experiment 1 and 2 no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

Additionally, in experiment 1 and 2 colony sizing showed no clastogenic effects induced by the test item under the experimental conditions.

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The mutagenic potential of Y-15099 was tested in bacteria according to the OECD guideline 471 (Koch, 2001). A plate incorporation test (Experiment 1) and a pre-incubation test (Experiment 2) using Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strain WP2 uvrA was performed in two independent experiments both with and without metabolic activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 0, 33, 100, 333, 1000, 2500, and 5000 µg/plate.


The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation. Toxic effects, evident as a reduction in number of revertants, were observed in all strains at concentrations of 1000 µg/plate or above. Additionally toxic effects were seen in strain TA100 at 100 µg/plate without S9 mix. No substantial increase in revertant colony numbers of any of the 5 tester strains was observed following treatment with Y-15099 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.


 


Y-15099, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster according to OECD guideline 473 (Koch, 2001). Two independent experiments, each with duplicates were performed. The highest applied concentration in the pre-test on toxicity (3650 µg/mL = 10 mM) was chosen with regard to the molecular weight of the test item and the current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of the test item insolubility in the culture medium. The chosen treatment concentrations were up to 50 µg/mL (Experiment 1 without metabolic activation) and up to 15 µg/mL (Experiment 2 without metabolic activation). With metabolic activation the chosen treatment concentrations were up to 160 µg/mL in both experiments. Per culture 100 metaphases were scored for structural chromosome aberrations.


Toxic effects indicated by reduced cell numbers and/or mitotic indices below 50% of the control values were observed in the absence of S9 mix only. In the presence of S9 mix no relevant toxicity was observed in the pre-test on toxicity. Therefore, the test item was tested up to a concentration exhibiting clear test item insolubility. In both independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens used as positive controls indicated the sensitivity of the test system. 


 


 


The read across substance Y-15821 (CAS 922519-17-3) was assessed in an in vitro Mammalian Cell Gene Mutation Test according to OECD 476 for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y (Kraft, 2008). The selection of test concentrations was based on data from the pre-experiment. In the first experiment 480 µg/mL (+S9) and 30 µg/mL (-S9) were selected as the highest concentrations. In the second experiment 515 µg/mL (+S9) and 39 µg/mL (-S9) were selected as the highest concentrations. Experiment 2 without metabolic activation was performed as 24 h long time exposure assay.


Growth inhibition was observed in experiment 1 and 2 with and without metabolic activation.


In experiment 1 with metabolic activation the relative total growth (RTG) was 19.17% for the highest concentration (480 µg/mL) evaluated. The highest biologically relevant concentration evaluated –S9 mix was 30 µg/mL with a RTG of 13.44%. In experiment 2 +S9 mix the RTG was 21.91% for the highest concentration (515 µg/mL) evaluated. The highest biologically relevant concentration evaluated – S9 mix was 39 µg/mL with a RTG of 12.93%.


No biologically relevant increase of mutants was found in any of the experimental settings. No dose-response relationship was observed. Additionally, in experiment 1 and 2 colony sizing showed no clastogenic effects induced by the test item under the experimental conditions, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.


 


Read-across justification for Y-15821 (CAS 922519-17-3):


The read across substance Y-15821 (CAS 922519-17-3) is a multi-component reaction mixture with variable composition. The variable composition results from the reaction of octanethioic acid, S-[3-(triethoxysilyl)propyl] ester (CAS 220727-26-4) with 2-methyl-1,3-propane diol (CAS 2163-42-0), and 3-(triethoxysilyl)-1-propanethiol (CAS 14814-09-6) to yield a product by process, partially oligomeric product mixture (CAS 922519-17-3).


The target substance Y-15099 (CAS 220727-26-4) is part of the multi-component reaction mixture Y-15821. Therefore, Y-15821 was taken as analogue substance into account by read-across.


Similarities of the read-across substance are reflected in the following comparable physico-chemical properties, toxicological characteristics and mode of action.


Y-15821 is also a liquid, which is poorly soluble in water and is subjected to quick hydrolysis. The vapour pressure is very low (0.00033 Pa at 25 °C; Smeykal, 2007). The read across substance has a boiling point of 241.9 °C at 101.3 kPa (Meinerling, 2007).


The acute toxicity of Y-15821 via the oral route is very low with an LD50 > 2000 mg/kg. This could be indicative of poor absorption through gastrointestinal tract. Y-15821 is virtually insoluble in water. This finding is in favour of assuming poor bioavailability of Y-15821. Y-15821 is also non-toxic via the percutaneous route. The MW of the major components is greater than 500 Da and the molecule is very lipophilic. Thus, systemic absorption through skin is expected to be low.


The major metabolic components of Y-15821 are esters of thiocarboxylic acids an as such subject to enzymatic and non-enzymatic hydrolysis yielding the respective diols and thioacids. Y-15821 has also been identified as a potent skin sensitizer. This indicates that the compound or its metabolites or hydrolysis products are capable of binding to proteins. It can further be assumed that the alkyl groups of Y-15821 will be subject to hydroxylation by hepatic monooxygenases if the substance enters portal blood at all. Y-15821 was negative in a murine micronucleus assay. This indicates that DNA-binding by the substance or its metabolites does not occur to a biologically relevant degree.


In terms of hazard assessment of toxic effects, available data on the genetic toxicity of Y-15821 was taken into account by read-across, since the target substance Y-15099 is a part of the reaction mixture.




Justification for classification or non-classification

According to CLP (1272/2008/EC) classification criteria for genetic toxicity, no classification is warranted.