Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2018

negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2018

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09-03-2018 to 10-04-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries (24 November 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017 ; signature: November 2017
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine or tryptophan locus
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Part of the first mutation test was repeated due to excessive toxicity (TA-strains dosed in the absence of S9-mix) employing an amended test item dose range of 0.015 to 50 µg/plate. Specifically: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and µg/plate.

Experiment 2 (pre-incubation method): Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item. The dose levels were selected based on the results of Experiment 1.

Salmonella strains (All); TA98, TA100, TA1535 and TA1537 (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate.
Salmonella strains TA100 and TA1537 (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strains TA1535 (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strain TA98 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
E.coli strain WP2uvrA (absence and presence of S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle.
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With metabolic activation S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1. in medium; in agar (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation).
The choice of application was due to the test item to either have unknown volatility or was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls.

DURATION
- Exposure duration:
Experiment 1. All of the plates were pre-incubated in sealed, small volume containers, by application of 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. All of the plates were sealed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure (37 ± 3 ºC for approximately 48 to 72 hours) to minimize potential losses of the test item from the plates. After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts may be performed, where automated counting cannot be performed: e.g. colonies spreading, colonies on the edge of the plates and artefacts on the plates, thus distorting the actual plate count.

Experiment 2. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media Subsequently, the procedure for incubation and duration was the same as in Experiment 1.

SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
In accordance with the OECD TG 471 guidelines.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.
Statistics:
Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.
Remarks on result:
other: all strains/cell types tested

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100 †

TA1535 †

WP2uvrA

TA98 †

TA1537 †

Solvent Control

(DMSO)

118

115

127

(120)

6.2#

11

20

12

(14)

4.9

33

18

27

(26)

7.5

22

19

22

(21)

1.7

16

17

5

(13)

6.7

0.015 µg

129

119

125

(124)

5.0

10

14

13

(12)

2.1

N/T

24

20

17

(20)

3.5

8

7

4

(6)

2.1

0.05 µg

130

128

115

(124)

8.1

18

7

14

(13)

5.6

N/T

14

14

18

(15)

2.3

8

5

7

(7)

1.5

0.15 µg

115

109

124

(116)

7.5

15

10

14

(13)

2.6

N/T

22

21

14

(19)

4.4

8

3

4

(5)

2.6

0.5 µg

96

116

123

(112)

14.0

8

8

10

(9)

1.2

N/T

19

17

26

(21)

4.7

3

4

4

(4)

0.6

1.5 µg

114

136

131

(127)

11.5

16

8

19

(14)

5.7

30

24

38

(31)

7.0

18

13

21

(17)

4.0

7

3

4

(5)

2.1

5 µg

105

93

100

(99)

6.0

12

11

11

(11)

0.6

25

28

32

(28)

3.5

16

19

17

(17)

1.5

7

8

7

(7)

0.6

15 µg

89 S

88 S

89 S

(89)

0.6

8 S

8 S

5 S

(7)

1.7

28

40

33

(34)

6.0

17 S

14 S

11 S

(14)

3.0

2 S

3 S

4 S

(3)

1.0

50 µg

0 V

0 V

0 V

(0)

0.0

6 S

8 S

12 S

(9)

3.1

40

39

36

(38)

2.1

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

150 µg

N/T

N/T

37

34

21

(31)

8.5

N/T

N/T

500 µg

N/T

N/T

31

32

26

(30)

3.2

N/T

N/T

1500 µg

N/T

N/T

29

22

24

(25)

3.6

N/T

N/T

5000 µg

N/T

N/T

34

46

19

(33)

13.5

N/T

N/T

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

564

352

374

(430)

116.6

173

201

213

(196)

20.5

549

522

443

(505)

55.1

324

336

341

(334)

8.7

162

86

224

(157)

69.1

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

137

136

131

(135)

3.2#

9

12

15

(12)

3.0

37

49

47

(44)

6.4

24

24

26

(25)

1.2

14

7

12

(11)

3.6

1.5 µg

131

134

128

(131)

3.0

14

8

16

(13)

4.2

49

32

34

(38)

9.3

24

19

22

(22)

2.5

7

10

12

(10)

2.5

5 µg

134

135

138

(136)

2.1

15

13

6

(11)

4.7

35

33

40

(36)

3.6

17

30

36

(28)

9.7

5

5

5

(5)

0.0

15 µg

128

129

123

(127)

3.2

11

10

15

(12)

2.6

34

39

39

(37)

2.9

32

28

27

(29)

2.6

8

13

6

(9)

3.6

50 µg

110

127

124

(120)

9.1

9

10

9

(9)

0.6

53

36

40

(43)

8.9

27

31

26

(28)

2.6

7

5

13

(8)

4.2

150 µg

74 S

93 S

77 S

(81)

10.2

15

12

12

(13)

1.7

29

50

48

(42)

11.6

31

21

28

(27)

5.1

4 S

9 S

10 S

(8)

3.2

500 µg

0 V

0 V

0 V

(0)

0.0

10 S

14 S

5 S

(10)

4.5

38

41

40

(40)

1.5

26

22

26

(25)

2.3

0 V

0 V

0 V

(0)

0.0

1500 µg

0 V

0 V

0 V

(0)

0.0

8 S

3 S

4 S

(5)

2.6

38

34

26

(33)

6.1

20

22

19

(20)

1.5

0 V

0 V

0 V

(0)

0.0

5000 µg

0 V

0 V

0 V

(0)

0.0

7 S

7 S

14 S

(9)

4.0

33

32

40

(35)

4.4

10 S

13 S

14 S

(12)

2.1

0 V

0 V

0 V

(0)

0.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1855

1919

2034

(1936)

90.7

333

319

289

(314)

22.5

229

231

232

(231)

1.5

105

126

146

(126)

20.5

346

364

367

(359)

11.4

†            Experimental procedure repeated at a later date due to toxicity in the original test

ENNG  N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO  4-Nitroquinoline-1-oxide

9AA      9-Aminoacridine

BP         Benzo(a)pyrene

2AA      2-Aminoanthracene

N/T      Not tested at this dose level

S            Sparse bacterial background lawn

V           Very weak bacterial background lawn

#           Standard deviation

 

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

102

114

115

(110)

7.2#

9

9

13

(10)

2.3

34

20

21

(25)

7.8

21

17

16

(18)

2.6

10

6

7

(8)

2.1

0.015 µg

107

115

126

(116)

9.5

8

9

9

(9)

0.6

N/T

15

25

18

(19)

5.1

9

6

7

(7)

1.5

0.05 µg

108

126

116

(117)

9.0

7

10

11

(9)

2.1

N/T

21

20

15

(19)

3.2

9

5

5

(6)

2.3

0.15 µg

117

133

118

(123)

9.0

14

10

6

(10)

4.0

N/T

20

13

26

(20)

6.5

11

12

8

(10)

2.1

0.5 µg

87

111

116

(105)

15.5

10

6

9

(8)

2.1

N/T

14

18

12

(15)

3.1

9

7

3

(6)

3.1

1.5 µg

123

123

128

(125)

2.9

7

8

13

(9)

3.2

N/T

15

19

15

(16)

2.3

6

5

3

(5)

1.5

5 µg

90

100

95

(95)

5.0

11

8

9

(9)

1.5

N/T

11

21

21

(18)

5.8

12

10

5

(9)

3.6

15 µg

82 S

87 S

83 S

(84)

2.6

12 S

15 S

7 S

(11)

4.0

28

29

23

(27)

3.2

22 S

18 S

18 S

(19)

2.3

6 S

5 S

5 S

(5)

0.6

50 µg

0 V

0 V

0 V

(0)

0.0

6 S

8 S

12 S

(9)

3.1

37

29

22

(29)

7.5

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

150 µg

N/T

N/T

31

25

21

(26)

5.0

N/T

N/T

500 µg

N/T

N/T

26

28

29

(28)

1.5

N/T

N/T

1500 µg

N/T

N/T

26

33

30

(30)

3.5

N/T

N/T

5000 µg

N/T

N/T

21

30

29

(27)

4.9

N/T

N/T

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

405

464

409

(426)

33.0

1268

1403

1552

(1408)

142.1

428

444

367

(413)

40.6

351

343

348

(347)

4.0

117

123

104

(115)

9.7

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

109

130

119

(119)

10.5#

11

13

19

(14)

4.2

41

32

35

(36)

4.6

29

23

31

(28)

4.2

10

10

15

(12)

2.9

0.15 µg

119

130

127

(125)

5.7

N/T

N/T

N/T

7

11

9

(9)

2.0

0.5 µg

112

110

113

(112)

1.5

8

10

12

(10)

2.0

N/T

N/T

12

7

8

(9)

2.6

1.5 µg

107

145

124

(125)

19.0

13

6

17

(12)

5.6

N/T

N/T

13

6

10

(10)

3.5

5 µg

120

129

131

(127)

5.9

14

10

7

(10)

3.5

N/T

27

21

24

(24)

3.0

4

13

5

(7)

4.9

15 µg

114

128

114

(119)

8.1

8

11

4

(8)

3.5

38

37

27

(34)

6.1

26

27

28

(27)

1.0

16

17

12

(15)

2.6

50 µg

104

124

106

(111)

11.0

12

12

10

(11)

1.2

41

38

33

(37)

4.0

27

19

23

(23)

4.0

12

8

11

(10)

2.1

150 µg

0 V

0 V

0 V

(0)

0.0

8

15

7

(10)

4.4

43

36

29

(36)

7.0

18

18

14

(17)

2.3

8 S

10 S

8 S

(9)

1.2

500 µg

0 V

0 V

0 V

(0)

0.0

8 S

7 S

9 S

(8)

1.0

39

29

31

(33)

5.3

13

18

27

(19)

7.1

0 V

0 V

0 V

(0)

0.0

1500 µg

N/T

0 V

0 V

0 V

(0)

0.0

30

31

41

(34)

6.1

20

10

11

(14)

5.5

N/T

5000 µg

N/T

N/T

39

26

51

(39)

12.5

10 S

17 S

27 S

(18)

8.5

N/T

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1863

1914

1789

(1855)

62.9

312

341

327

(327)

14.5

162

177

235

(191)

38.6

104

106

132

(114)

15.6

275

233

250

(253)

21.1

Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item using the Ames pre incubation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. Part of the first mutation test was repeated due to excessive toxicity (all TA-strains) dosed in the absence of S9-mix employing an amended test item dose range of 0.015 to 50 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again selected in Experiment 2 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range was amended following the results of Experiment 1 and ranged between 0.015 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit of the test item depending on the strain type and presence of S9-mix.The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in the absence of S9-mix from 15 μg/plate. In the presence S9-mix, weakened bacterial background lawns were notedfor all of the Salmonella strains initially from 150 μg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence S9-mix. In Experiment 2, both the maximum dose level (5000 μg/plate) or the toxic limit was employed as the maximum concentration in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item induced an identical toxic response to the first experiment with weakened bacterial background lawns noted from 15 μg/plate to all Salmonella strains dosed in the absence and presence of S9-mix. Again, no toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level. No precipitates were observed at any dose level in either the presence or absence of S9-mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-04-2018 to 27-06-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
: Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017 ; signature: November 2017
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable (chromosome aberration test)
Species / strain / cell type:
lymphocytes: Human lymphocytes
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours. Further details on the donors is available in the full study report.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction: Lot No. PB/βNF S9 29/03/2018
Test concentrations with justification for top dose:
The maximum dose level was 1940 µg/mL, calculated to be equivalent to 10 mM concentration, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1940 μg/mL range (full results recorded in the full study report). Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details.

I. Preliminary toxicity test: 0 (control) , 7.6, 15.2, 30.3, 60.6, 121.3, 242.5, 485, 970 and 1940 μg/mL
Within three exposure groups:
i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

II. Main Test:
4(20)-hour without S9: 0*, 10, 15*, 30*, 60*, 80, 120, 240 μg/mL and MMC 0.4* μg/mL
4(20)-hour with S9: 0*, 10, 15, 30*, 60*, 80*, 120, 240, μg/mL and CP 4* μg/mL
24-hour without S9: 0*, 10, 20, 30, 40*, 50*, 60*, 120 μg/mL and MMC 0.1* μg/mL
where:
* = dose levels selected for metaphase analysis
MMC= Mitomycin C
CP = Cyclophosphamide
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In a previously conducted OECD TG 471 study (cited in the full study report), the test item was immiscible in aqueous media at 50 mg/mL but was miscible in dimethyl sulphoxide at the same concentration. A further solubility check was performed within this study. Dimethyl sulphoxide and acetone indicated that the test item was miscible at 194 mg/mL. Dimethyl sulphoxide was chosen due to better suitability when used with human lymphocytes. The maximum dose level (determined prior to the test based on molecular weight) was 1940 µg/mL, which was calculated to be equivalent to 10 mM, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1940 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system.
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details
Untreated negative controls:
other: Vehicle control served as the negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation, 0.4 μg/ml for 4(20)-hour and 0.1 μg/ml for 24 h exposure periods, respectively ; Full details on the positive controls is reported in the full study report.
Untreated negative controls:
other: Vehicle control served athe negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation 4 μg/ml for a 4(20)-hour exposure period ; Full details on the positive controls is reported in the full study report.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Other:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile non-vented plastic flasks for each culture: 8.05 to 9.05 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood

DURATION
- Preincubation period: Not reported.
- Exposure duration:
The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below) in the absence of metabolic activation only.
I. With Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL (100 μL) of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of the Main Experiment. After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.

II. Without Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL (100 μL) of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
In the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 μg/ml)

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

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

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range was reported in the full study report.
- Other: Scoring: Where possible, 300 consecutive well-spread metaphases from each concentration (150 per duplicate) were assessed for observations, if the cell had 44 to 48 chromosomes, any breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations according to the simplified system of Savage (1976), ISCN (1985). Where the analysis of the slide resulted in a large frequency of aberrant cells then the analysis was terminated after a total of 15 metaphases with aberrations (excluding gaps) were recorded. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
Evaluation criteria:
Positive response criteria
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.

Negative response criteria
A test item can be classified as non-genotoxic if:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.

In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment.

Statistical analysis is also performed (see: ‘Statistics’). Biological relevance of the results are to be considered first. Statistical methods are used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response. A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: There was no significant change osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported. Sterile non-vented plastic flasks for each culture, thereby preventing evaporation from medium.
- Water solubility: Not applicable.
- Precipitation: In the preliminary test: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 121.3 µg/mL in all of the exposure groups.
Main test: Precipitate observations were made at the end of exposure in blood-free cultures and was noted at 240 μg/mL in the 4(20)-hour exposure group dosed in the absence of S9 and at and above 120 μg/mL in the presence of S9 and 24-hour exposure group. The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present at up 60 and 80 μg/mL of test item in the 4(20)-hour exposure groups and up to 60 µg/mL in the 24-hour continuous exposure group.
- Other confounding effects: In the preliminary test: Hemolysis was observed following exposure to the test item at and above 60.6 μg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9), at and above 30.3 μg/mL in the 4(20)-hour in the presence of S9 and at and above 121.3 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. Additionally, a reduced cell pellet was observed at and above 121.3 μg/mL in the 4(20)-hour exposure groups and at and above 242.5 μg/mL in the 24-hour exposure group. A reduced pellet signifies that maximum exposure is occurring in the remaining cell population. In the main test: Hemolysis was again observed in the Main Experiment at and above 30 μg/mL in the in the presence of S9 and at and above 60 μg/mL in the absence of S9. Additionally, a reduced cell pellet was observed at and above 80 μg/mL in the short exposure groups only.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1940 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity for the main test. Specifically based on precipitation and cell cytotoxicity. 60 to 80 μg/mL in the 4(20)-hour exposure groups and up to 60 µg/mL in the 24-hour continuous exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations. (statistically significant p ≤ 0.01 or were within the Historic Control Data range presented in the full study report).

ADDITIONAL INFORMATION ON CYTOTOXICITY: See ‘other confounding effects’ listed above.

1. Chromosome Aberration Test – Main Test

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

4(20)-hour without S9: 0*, 10, 15*, 30*, 60*, 80, 120, 240 μg/mL and MMC 0.4* μg/mL

4(20)-hour with S9: 0*, 10, 15, 30*, 60*, 80*, 120, 240, μg/mL and CP 4* μg/mL

24-hour without S9: 0*, 10, 20, 30, 40*, 50*, 60*, 120 μg/mL and MMC 0.1* μg/mL

where: * = dose levels selected for metaphase analysis ; MMC= Mitomycin C and CP = Cyclophosphamide

 

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present at up 60 and 80 μg/mL of test item in the 4(20)-hour exposure groups and up to 60 µg/mL in the 24-hour continuous exposure group. Precipitate observations were made at the end of exposure in blood-free cultures and was noted at 240 μg/mL in the 4(20)-hour exposure group dosed in the absence of S9 and at and above 120 μg/mL in the presence of S9 and 24-hour exposure group. Additionally, hemolysis was observed following exposure to the test item at and above 60.6 μg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9), at and above 30.3 μg/mL in the 4(20)-hour in the presence of S9 and at and above 121.3 μg/mL in the 24-hour continuous exposure group. A reduced cell pellet was observed at and above 80 μg/mL in the short exposure groups 4(20)-hours, only.

The qualitative observations indicated a dose-related inhibition of mitotic index was observed in all exposure groups.In the 4(20)-hour exposure group in the absence of S9, 70% mitotic inhibition was achieved at 60 μg/mL. Below this dose level no inhibition was observed; above it there were no metaphases present. Therefore, the maximum dose level selected for metaphase analysis was 60 μg/mL, even though it exceeded the maximum range for optimum toxicity.In the presence of S9, 32%, 23% and 55% mitotic inhibition was achieved at 30, 60 and 80 μg/mL, respectively. Above this dose level, there were no scorable metaphases present. Therefore, the maximum dose level selected for metaphase analysis was 80 μg/mL because this dose level achieved optimum toxicity as defined by the OECD 473 test guideline (55±5%). In the 24-hour continuous exposure group, an inhibition of mitotic index of 40% and 58% was noted at 50 and 60 μg/mL, respectively. The maximum dose level selected for metaphase analysis was, therefore, 60 μg/mL because this dose level achieved optimum toxicity as defined by the OECD 473 test guideline (55±5%).

 

- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.

- The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

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

- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. There were several indications of endoreduplication noted, each isolated within each exposure group. The applicant confirms: endoreduplicated cell incidences were not replicated in the duplicate cultures at any dose-level.

 

2. Discussion:

- In the 4(20)-hour exposure group in the absence of S9, 70% mitotic inhibition was achieved at 60 μg/mL. Below this dose level no inhibition was observed; above it there were no metaphases present. Therefore, the maximum dose level selected for metaphase analysis was 60 μg/mL, even though it exceeded the maximum range for optimum toxicity.Below this dose level (30%) no inhibition was observed but above it (80 μg/mL) there were no metaphases present. Despite this, the test item did not induce any statistically significant increases in the frequency of cells with aberrations, even though the dose level selected could be considered excessively toxic. The hemolysis and reduced pellet observations for this exposure group support that the lymphocytes were at or near to maximum exposure of the test item.

- In the 4(20)-hour exposure group in the presence of S9, 32%, 23% and 55% mitotic inhibition was achieved at 30, 60 and 80 μg/mL, respectively. Above this dose level, there were no scorable metaphases present. Therefore, the maximum dose level selected for metaphase analysis was 80 μg/mL because this dose level achieved optimum toxicity as defined by the OECD 473 test guideline (55±5%). The hemolysis and reduced pellet observations for this exposure group support that the lymphocytes were at or near to maximum exposure of the test item.

- In the 24-hour exposure group an inhibition of mitotic index of 40% and 58% was noted at 50 and 60 μg/mL, respectively. The maximum dose level selected for metaphase analysis was, therefore, 60 μg/mL because this dose level achieved optimum toxicity as defined by the OECD 473 test guideline (55±5%).

- Optimum toxicity or greater than optimum toxicity was achieved in the cultures in the absence of S9 and presence of S9. Under the conditions of the study, the test item was considered to have been adequately tested as the test item was tested up to cytotoxic conditions and scored at dose levels within or above, excessive cytotoxicity. No significant increases in chromosome aberrations was observed at any dose level or exposure group tested.

Conclusions:
Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The study was performed to the requirements of OECD TG 473 and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test (Cell Growth Inhibition Test) between 7.6 to 1940 μg/mLwhere the results indicated that the maximum concentration should be limited on precipitation and toxicity for all exposure groups for dose selection. Precipitation and toxicity closely coincided. The dose levels selected for the Main Test were as follows: 4(20)-hour with and without S9-Mix (2%) : 0, 10, 15, 30, 60, 80, 120, 240 μg/mL and 24-hour continuous exposure without S9: 0, 10, 20, 30, 40, 50, 60, 120 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item was toxic to human lymphocytes and but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that either induced or exceeded 55±5% mitotic inhibition. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key study : OECD TG 471, 2018 : The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item using the Ames pre incubation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. Part of the first mutation test was repeated due to excessive toxicity (all TA-strains) dosed in the absence of S9-mix employing an amended test item dose range of 0.015 to 50 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again selected in Experiment 2 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range was amended following the results of Experiment 1 and ranged between 0.015 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit of the test item depending on the strain type and presence of S9-mix.The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in the absence of S9-mix from 15 μg/plate. In the presence S9-mix, weakened bacterial background lawns were notedfor all of the Salmonella strains initially from 150 μg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence S9-mix. In Experiment 2, both the maximum dose level (5000 μg/plate) or the toxic limit was employed as the maximum concentration in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item induced an identical toxic response to the first experiment with weakened bacterial background lawns noted from 15 μg/plate to all Salmonella strains dosed in the absence and presence of S9-mix. Again, no toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level. No precipitates were observed at any dose level in either the presence or absence of S9-mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Key study : OECD TG 473, 2018 : The study was performed to the requirements of OECD TG 473 and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test (Cell Growth Inhibition Test) between 7.6 to 1940 μg/mLwhere the results indicated that the maximum concentration should be limited on precipitation and toxicity for all exposure groups for dose selection. Precipitation and toxicity closely coincided. The dose levels selected for the Main Test were as follows: 4(20)-hour with and without S9-Mix (2%) : 0, 10, 15, 30, 60, 80, 120, 240 μg/mL and 24-hour continuous exposure without S9: 0, 10, 20, 30, 40, 50, 60, 120 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item was toxic to human lymphocytes and but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that either induced or exceeded 55±5% mitotic inhibition. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity