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, 2019

negative, in vitro mammalian cell micronucleus assay (with and without S-9 activation), human lymphocytes, OECD TG 487, 2020

Link to relevant study records
Reference
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:
26-06-2019 to 19-07-2019
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
Qualifier:
according to
Guideline:
other: ICH S2(R1) guideline
Version / remarks:
June 2012 (ICH S2(R1) Federal Register
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: August 2018 ; signature: November 2018
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:
Type and composition of metabolic activation system: Rat liver S9
- source of S9: Purchased from recognised supplier (dates within full study report) ; S9 Microsomal fraction: Lot No. Lot No. 4061
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 QC and Production Certificate including Activity is presented in the full study report. Additionally, concurrent positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory
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
Experiment 2 (pre-incubation method): Eight to ten 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.
All strains (absence of S9-mix): 0.5, 1.5, 5, 15, 50, 75, 100, 150 µg/plate.
All strains (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 75, 100, 125, 150, 175, 500 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulphoxide (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 DMSO at the same concentration in solubility checks performed. DMSO 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.
Key result
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
Key result
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.

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)

108

07

98

(104)

5.5#

15

25

16

(19)

5.5

28

31

27

(29)

2.1

15

20

16

(17)

2.6

9

11

11

(10)

1.2

1.5 µg

115

110

103

(112)

2.9

20

14

26

(20)

6.0

35

28

21

(28)

7.0

18

19

15

(17)

2.1

9

6

9

(8)

1.7

5 µg

103

122

107

(111)

10.0

15

15

16

(15)

0.6

26

37

27

(30)

6.1

9

19

20

(6)

6.1

11

14

8

(11)

3.0

15 µg

100

119

119

(113)

11.0

29

13

15

(19)

8.7

25

24

35

(28)

6.1

14

14

18

(15)

2.3

10

6

17

(11)

5.6

50 µg

126

163

165

(151)

22.0

18

11

19

(16)

4.4

33

19

37

(30)

9.5

11

21

17

(16)

5.0

8

17

12

(12)

4.5

150 µg

0 V

0 V

0 V

(0)

0.0

15 S

14 S

12 S

(14)

1.5

25 S 29 S

21 S

(25)

4.0

8 S

15 S

9 S

(11)

3.8

10 S

6 S

5 S

(7)

2.6

500 µg

0 V

0 V

0 V

(0)

0.0

8 S

21 S

23 S

(17)

8.1

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

11 S

5 S

5 S

(7)

3.5

1500 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

5000 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

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

588

538

571

(566)

25.4

1337

1433

1383

(1384)

48.0

514

527

478

(506)

25.4

191

182

187

(187)

4.5

181

179

248

(203)

39.3

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)

96

109

104

(103)

6.6#

11

15

11

(12)

2.3

47

35

28

(37)

9.6

20

18

20

(19)

1.2

15

15

17

(16)

1.2

1.5 µg

103

91

98

(97)

6.0

11

14

15

(13)

2.1

40

35

30

(35)

5.0

18

18

24

(20)

6.7

11

12

12

(12)

0.6

5 µg

99

116

107

(107)

8.5

9

11

16

(12)

3.6

30

37

37

(35)

4.0

27

15

16

(19)

6.7

13

11

15

(13)

2.0

15 µg

102

115

103

(107)

7.2

14

7

7

(9)

4.0

41

37

49

(42)

6.1

22

36

22

(27)

8.1

20

14

10

(15)

5.0

50 µg

100

101

101

(101)

0.6

20

16

13

(16)

3.5

32

45

32

(36)

7.5

23

17

22

(21)

3.2

11

13

8

(11)

2.5

150 µg

208 S

227 S

121 S

(185)

56.5

10

9

4

(8)

3.2

43

32

29

(35)

7.4

17

19

20

(19)

1.5

7

10

11

(9)

2.1

500 µg

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

15 S

21 S

17 S

(18)

3.1

0 V

0 V

0 V

(0)

0.0

1500 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

5000 µg

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(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

1263

964

1024

(1084)

158.2

234

230

235

(233)

2.6

148

139

132

(140)

8.0

112

139

121

(124)

13.7

234

205

220

(220)

14.5

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

T            Toxic, no 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 #2

TA1537

Solvent Control

(DMSO)

112

124

110

(115)

7.60#

13

10

13

(12)

1.7

22

21

24

(22)

1.5

18

23

17

(19)

3.2

6

10

14

(10)

4.0

0.5 µg

114

112

116

(114)

2.0

14

12

10

(12)

2.0

20

19

25

(21)

3.2

6

19

15

(13)

6.7

10

5

7

(7)

2.5

1.5 µg

124

122

107

(118)

9.3

21

9

13

(14)

6.1

20

18

20

(19)

1.2

17

18

24

(20)

3.8

11

10

14

(12)

2.1

5 µg

109

108

129

(115)

11.8

9

16

18

(14)

4.7

26

22

23

(24)

2.1

13

17

17

(16)

2.3

9

9

8

(9)

0.6

15 µg

115

118

113

(115)

2.5

11

10

9

(10)

1.0

21

19

17

(19)

2.0

13

19

22

(19)

4.6

15

11

13

(13)

2.0

50 µg

99 S

107 S

110 S

(105)

5.7

16

10

12

(13)

3.1

18

21

24

(21)

3.0

22

12

15

(16)

5.1

9

15

11

(12)

3.1

75 µg

111 S

95 S

114 S

(107)

10.2

0 V

0 V

0 V

(0)

0.0

15

17

20

(17)

2.5

12

26

14

(17)

7.6

9

13

7

(10)

3.1

100 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

25 S

20 S

16 S

(20)

4.5

19 S

11 S

13 S

(14)

4.2

7 S

9 S

3 S

(6)

3.1

150 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

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

475

561

596

(544)

62.3

1098

1229

1410

(1246)

156.7

331

338

368

(346)

19.7

147

162

125

(145)

18.6

177

160

171

(169)

8.6

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98 #2

TA1537

Solvent Control

(DMSO)

120

110

107

(112)

6.8#

9

12

12

(11)

1.7

25

24

25

(25)

0.6

29

26

18

(24)

5.7

12

10

15

(12)

2.5

0.5 µg

86

95

101

(94)

7.5

11

10

12

(11)

1.0

33

27

30

(30)

3.0

23

27

23

(24)

2.3

8

6

13

(9)

3.6

1.5 µg

108

130

116

(118)

11.1

14

7

10

(10)

3.5

30

26

19

(25)

5.6

22

27

27

(25)

2.9

11

11

14

(12)

1.7

5 µg

121

115

102

(113)

9.7

9

13

14

(12)

2.6

21

36

34

(30)

8.1

26

24

22

(24)

2.0

9

12

12

(11)

1.7

15 µg

117

104

109

(110)

6.6

14

12

7

(11)

3.6

33

32

34

(33)

1.0

22

23

15

(20)

4.4

15

8

12

(12)

3.5

50 µg

101

108

96

(102)

6.0

6

9

15

(10)

4.6

30

18

35

(28)

8.7

32

24

32

(29)

4.6

14

19

4

(12)

7.6

100 µg

99 S

108 S

109 S

(105)

5.5

11

8

19

(13)

5.7

34

34

28

(32)

3.5

20

18

20

(19)

1.2

12

7

6

(8)

3.2

125 µg

0 V

0 V

0 V

(0)

0.0

14 S

8 S

12 S

(11)

3.1

30

18

28

(25)

6.4

34

23

18

(25)

8.2

16

12

9

(12)

3.5

150 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

25 S

41 S

30 S

(32)

8.2

16

20

15

(17)

2.6

11

19

10

(13)

4.9

175 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

31 S

24 S

38 S

(31)

7.0

24 S

17 S

23 S

(21)

3.8

7 S

12 S

15 S

(11)

4.0

500 µg

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(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

1471

1326

1373

(1390)

74.0

236

223

241

(233)

9.3

103

112

127

(114)

12.1

120

123

100

(114)

12.5

194

163

130

(162)

32.0

#2 : Experimental procedure repeated at a later date due to contamination in the original test

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 ten 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. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Up to ten test item dose levels were 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 0.5 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (DMSO) 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 maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 μg/plate in both the absence and presence of metabolic activation (S9-mix). Based on the results of Experiment 1, the toxic limit was employed as the maximum concentration in the second mutation test. The test item once again induced a toxic response with a visible reduction in the growth of the bacterial background lawns noted for all of the tester strains, initially from 50 and 100 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively. There were no biologically relevant 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 (S9-mix) in Experiment 1. One statistically significant value was noted (TA100 at 50 μg/plate in the absence of metabolic activation (S9-mix). However, this response was neither dose-related nor reproducible, and was within the in-house historical vehicle/untreated control range for the strain and, therefore considered of no biological relevance. A further statistical value was also noted (TA100 at 150 μg/plate in the presence of metabolic activation (S9-mix)), however this concentration exhibited toxicity as weakened bacterial background lawns therefore nullifying the statistical value.No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. No precipitates were observed at any dose level in either the presence or absence of S9-mix. There were no biologically relevant 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 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, 2019 : 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 ten 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. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Up to ten test item dose levels were 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 0.5 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (DMSO) 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 maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 150 μg/plate in both the absence and presence of metabolic activation (S9-mix). Based on the results of Experiment 1, the toxic limit was employed as the maximum concentration in the second mutation test. The test item once again induced a toxic response with a visible reduction in the growth of the bacterial background lawns noted for all of the tester strains, initially from 50 and 100 μg/plate in the absence and presence of metabolic activation (S9-mix), respectively. There were no biologically relevant 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 (S9-mix) in Experiment 1. One statistically significant value was noted (TA100 at 50 μg/plate in the absence of metabolic activation (S9-mix). However, this response was neither dose-related nor reproducible, and was within the in-house historical vehicle/untreated control range for the strain and, therefore considered of no biological relevance. A further statistical value was also noted (TA100 at 150 μg/plate in the presence of metabolic activation (S9-mix)), however this concentration exhibited toxicity as weakened bacterial background lawns therefore nullifying the statistical value.No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. No precipitates were observed at any dose level in either the presence or absence of S9-mix. There were no biologically relevant 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 487, 2020 : The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the test item ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (1.8% S9-mix). The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments. In a preliminary dose-range finding test at : 0 (control), 63,125, 250, 500, 1000, 1783 μg/mL (i.e. up to 0.01M or 10 mM) the cytotoxicity of the test item to the human lymphocytes was assessed using the cytokinesis-block proliferation index (CBPI index). The vehicle utilised was dimethylsulphoxide (DMSO). Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control. In the first cytogenetic assay, the test item was tested up to 175 µg/mL and 225 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Appropriate toxicity was reached at these dose levels with % cytostasis: 57% and 66%, at 175 µg/mL (-S9 fraction) and 225 µg/mL (+S9 fraction), respectively. In the second cytogenetic assay, the test item was tested up to 40 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels with % cytostasis: 58% at 40 µg/mL (-S9 fraction). The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei in the (3)-27 hours and (24)-24 hours exposure and harvest times and the absence S9-fraction. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei in the (3)-27 hours exposure and harvest time and presence of S9-fraction. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic 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