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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 20 September 2017 Experimental completion date: 23 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
Batch: 52611021
CAS Number: 68475-94-5
EC Number: 500-215-4
Purity: 95-100%
Physical state / Appearance: Clear yellow liquid
Expiry Date: 01 December 2018
Storage Conditions: Room temperature in the dark
Specific details on test material used for the study:
Identification: Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin
CAS Number: 68475-94-5
Physical state/Appearance: Clear yellow liquid
Batch: 52611021
Purity: 100% (UVCB product)
Expiry Date: 12 January 2018
Storage Conditions: Room temperature in the dark

Method

Target gene:
histidine locus in S. typhimurium and tryptophan locus in E. coli.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
10% rat liver S9 in standard co-factors
Test concentrations with justification for top dose:
Experiment 1 - Plate Incorporation Method
The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, and 5000 µg/plate.
Vehicle / solvent:
The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in house. Acetone was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
80 µg/plate for TA1537
Positive control substance:
9-aminoacridine
Remarks:
absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/plate for TA98
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
presence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate for TA98
Positive control substance:
benzo(a)pyrene
Remarks:
presence of S9-mix
Details on test system and experimental conditions:
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were required for verification due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre incubation method in the presence and absence of metabolic activation.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent 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, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Some manual counts were performed due to spreading colonies to ensure an accurate count.

Test for Mutagenicity: Confirmatory Experiment – Pre-Incubation and Plate Incorporation Methods
As Experiment 2 was concluded to be positive in TA1535 only, a third, confirmatory experiment was performed using both the pre incubation and plate incorporation methods in the presence and absence of metabolic activation.
The dose range was determined by the results of Experiment 2 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.

Without Metabolic Activation
Performed as described in plate incorporation method.

With Metabolic Activation
Performed as described in plate incorporation method

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

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. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. 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)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (acetone) control plates gave counts of revertant colonies generally within the normal range. Some of the vehicle/untreated control counts for TA1535 and TA98 in the first mutation test were just above the in-house control maxima. These counts were still considered acceptable as the majority of counts were within the historical profile and all were within the expected control range outlined for the strains in the GSP and the tester strains responded very well with the respective positive controls in both the presence and absence of S9 mix.
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.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No toxicity was observed in the confirmatory experiment at any dose level, either in the presence or absence of metabolic activation (S9-mix).

A greasy test item precipitate was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate.
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 (plate incorporation method). Small increases were noted in Experiment 1 (WP2uvrA at 150 and 5000 µg/plate in the absence of S9-mix), however these responses were within the in-house historical vehicle/untreated control values for the strain and were, therefore considered of no biological relevance.
In light of these results, Experiment 2 was conducted using the pre-incubation method.

Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
Dose-related and statistically significant increases in TA1535 revertant colony frequency were observed at and above 500 µg/plate in the absence of S9-mix and from 150 µg/plate in the presence of S9-mix. In the absence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 1500 µg/plate with a maximum fold increase over the concurrent vehicle control of 9.3 at 5000 µg/plate. In the presence of S9, the mean colony counts were just in excess of the in-house upper historical control maxima from 150 µg/plate with a maximum fold increase over the concurrent vehicle control of 13.1 noted at 5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded for any of the remaining Salmonella or Escherichia coli bacterial strains, with any dose of the test item, either with or without metabolic activation.

Experiment 3 (Confirmatory Test)
In light of the discrepancy in results between Experiments 1 and 2, a third test was conducted employing both the pre-incubation and plate incorporation methods and a single strain of Salmonella typhimurium, TA1535 in the absence and presence of S9-mix.
In the pre-incubation, statistically significant increases in TA1535 revertant colony frequency were observed, initially from 150 µg/plate in the absence of S9-mix and 50 µg/plate in the presence of S9-mix. In the absence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 1500 µg/plate with a maximum fold increase over the concurrent vehicle control of 6.5 at 5000 µg/plate. In the presence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 150 µg/plate with a maximum fold increase over the concurrent vehicle control of 12 noted at 5000 µg/plate.
In the plate incorporation assay, statistically significant increases in TA1535 revertant colony frequency were also observed initially from 50 µg/plate in the absence of S9-mix and 15 µg/plate in the presence of S9-mix. In the absence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 500 µg/plate with a maximum fold increase over the concurrent vehicle control of 11.9 at 5000 µg/plate. In the presence of S9, the mean colony counts were in excess of the in-house upper historical control maxima from 50 µg/plate with a maximum fold increase over the concurrent vehicle control of 21.1 noted at 5000 µg/plate.

Any other information on results incl. tables

Spontaneous Mutation Rates (ConcurrentNegativeControls)Experiment1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

95

 

30

 

22

 

49

 

18

 

120

(111)

38

(34)

29

(25)

51

(51)

21

(20)

119

 

35

 

23

 

52

 

22

 

 

Experiment 2

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

111

 

13

 

22

 

19

 

14

 

110

(114)

17

(15)

24

(26)

18

(19)

19

(17)

122

 

14

 

32

 

21

 

19

 

 

Experiment 3 – Confirmatory Test: Plate incorporation

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

TA1535

13

 

11

(12)

11

 

 

Experiment 3 – Confirmatory Test: Pre-Incubation

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

TA1535

12

 

17

(15)

15

 

Test Results: Experiment 1 – Without MetabolicActivation(Plate Incorporation)

 

Test Period

From: 06 October 2017

To: 09 October 2017

 

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

125

109

116

(117)

8.0#

32

30

34

(32)

2.0

31

26

19

(25)

6.0

47

43

45

(45)

2.0

14

12

11

(12)

1.5

 

1.5 µg

130

107

109

(115)

12.7

33

32

41

(35)

4.9

28

29

23

(27)

3.2

44

39

52

(45)

6.6

9

13

10

(11)

2.1

 

5 µg

112

119

120

(117)

4.4

29

27

31

(29)

2.0

24

23

24

(24)

0.6

50

46

48

(48)

2.0

11

11

15

(12)

2.3

 

15 µg

117

103

124

(115)

10.7

31

31

38

(33)

4.0

28

29

30

(29)

1.0

43

39

55

(46)

8.3

10

8

14

(11)

3.1

 

50 µg

137

133

126

(132)

5.6

36

34

36

(35)

1.2

22

27

20

(23)

3.6

49

41

40

(43)

4.9

12

13

11

(12)

1.0

 

150 µg

98

121

114

(111)

11.8

40

29

32

(34)

5.7

34

29

35

(33)

3.2

52

52

40

(48)

6.9

10

8

15

(11)

3.6

 

500 µg

93

134

116

(114)

20.6

33

32

30

(32)

1.5

29

26

31

(29)

2.5

38

44

44

(42)

3.5

11

7

9

(9)

2.0

 

1500 µg

80 P

104 P

85 P

(90)

12.7

29 P

35P

36P

(33)

3.8

35 P

33 P

26 P

(31)

4.7

49 P

51 P

47 P

(49)

2.0

10 P

13 P

12 P

(12)

1.5

 

5000 µg

111 P

98 P

103 P

(104)

6.6

33 P

35 P

31 P

(33)

2.0

35 P

31 P

33 P

(33)

2.0

45 P

51 P

49 P

(48)

3.1

11 P

11 P

8 P

(10)

1.7

Positive controls S9-Mix (-)

 

Name DoseLevel

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

598

608

524

(577)

45.9

923

1115

952

(997)

103.5

871

964

1029

(955)

79.4

178

214

216

(203)

21.4

235

393

309

(312)

79.1

 

 

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

4NQO       4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

P           Test itemprecipitate

#            Standarddeviation


 

Test Results: Experiment 1 – With MetabolicActivation(Plate Incorporation)

 

Test Period

From: 06 October 2017

To: 09 October 2017

 

 

 

 

 

 

 

 

 

 

 

 

 

S9-Mix (+)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

91

122

117

(110)

16.6#

38

36

39

(38)

1.5

40

34

38

(37)

3.1

51

53

50

(51)

1.5

17

15

22

(18)

3.6

 

1.5 µg

113

115

107

(112)

4.2

41

40

43

(41)

1.5

32

34

32

(33)

1.2

46

53

51

(50)

3.6

19

17

12

(16)

3.6

 

5 µg

102

123

115

(113)

10.6

39

44

38

(40)

3.2

41

45

45

(44)

2.3

50

57

55

(54)

3.6

16

15

19

(17)

2.1

 

15 µg

117

116

110

(114)

3.8

38

37

38

(38)

0.6

28

41

40

(36)

7.2

55

53

51

(53)

2.0

19

20

22

(20)

1.5

 

50 µg

104

119

98

(107)

10.8

42

43

40

(42)

1.5

34

36

34

(35)

1.2

48

58

52

(53)

5.0

21

20

16

(19)

2.6

 

150 µg

105

112

89

(102)

11.8

41

39

39

(40)

1.2

34

37

36

(36)

1.5

52

50

56

(53)

3.1

15

18

19

(17)

2.1

 

500 µg

112

106

101

(106)

5.5

43

45

39

(42)

3.1

33

26

30

(30)

3.5

52

49

57

(53)

4.0

18

19

20

(19)

1.0

 

1500 µg

99 P

101 P

105 P

(102)

3.1

42 P

46 P

41 P

(43)

2.6

35 P

36 P

30 P

(34)

3.2

51 P

51 P

54 P

(52)

1.7

22 P

17 P

19 P

(19)

2.5

 

5000 µg

105 P

110 P

122 P

(112)

8.7

47 P

37P

38P

(41)

5.5

39 P

41 P

40 P

(40)

1.0

55 P

56 P

50 P

(54)

3.2

22 P

24 P

17 P

(21)

3.6

Positive controls S9-Mix (+)

 

Name DoseLevel

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2368

2524

2509

(2467)

86.1

298

302

281

(294)

11.2

542

492

547

(527)

30.4

285

450

454

(396)

96.4

394

376

296

(355)

52.2

 

 

BP         Benzo(a)pyrene

2AA    2-Aminoanthracene

P      Test item precipitate

#            Standard deviation

Test Results: Experiment 2 – Without MetabolicActivation(Pre- Incubation)

 

Test Period

From: 13 October 2017

To: 16 October 2017

 

 

 

 

 

 

 

 

 

 

S9-Mix (-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

111

118

104

(111)

7.0#

11

11

21

(14)

5.8

29

26

23

(26)

3.0

15

25

15

(18)

5.8

18

18

18

(18)

0.0

 

15 µg

140

105

111

(119)

18.7

9

12

11

(11)

1.5

35

28

36

(33)

4.4

21

5

18

(15)

8.5

9

26

27

(21)

10.1

 

50 µg

88

99

99

(95)

6.4

14

10

14

(13)

2.3

30

23

23

(25)

4.0

21

23

19

(21)

2.0

28

19

21

(23)

4.7

 

150 µg

109

136

94

(113)

21.3

15

22

19

(19)

3.5

19

21

24

(21)

2.5

21

23

27

(24)

3.1

16

18

14

(16)

2.0

 

500 µg

103

124

121

(116)

11.4

25

35

25

** (28)

5.8

26

28

23

(26)

2.5

33

25

17

(25)

8.0

18

26

15

(20)

5.7

 

1500 µg

127 P

132 P

104 P

(121)

14.9

54 P

68 P

65 P

*** (62)

7.4

32 P

40 P

29 P

(34)

5.7

20 P

31 P

29 P

(27)

5.9

16 P

33 P

21 P

(23)

8.7

 

5000 µg

111 P

126 P

142 P

(126)

15.5

152 P

123 P

115 P

*** (130)

19.5

34 P

37 P

27 P

(33)

5.1

18 P

17 P

16 P

(17)

1.0

22 P

22 P

29 P

(24)

4.0

Positive controls S9-Mix (-)

 

Name DoseLevel

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

1072

934

986

(997)

69.7

1278

1235

1592

(1368)

194.9

882

859

955

(899)

50.1

306

300

320

(309)

10.3

278

246

315

(280)

34.5

 

 

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

4NQO      4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

P             Test item precipitate

**             p0.01

***           p0.00

#                Standard deviation


 

Test Results: Experiment 2 – With MetabolicActivation(Pre- Incubation)

 

Test Period

From: 13 October 2017

To: 16 October 2017

 

 

 

 

 

 

 

 

 

 

S9-Mix (+)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (Acetone)

111

88

98

(99)

11.5#

9

20

12

(14)

5.7

40

32

30

(34)

5.3

30

30

24

(28)

3.5

20

21

14

(18)

3.8

 

15 µg

127

94

85

(102)

22.1

22

3

13

(13)

9.5

39

25

32

(32)

7.0

17

26

26

(23)

5.2

10

17

24

(17)

7.0

 

50 µg

93

106

110

(103)

8.9

28

34

22

(28)

6.0

36

31

44

(37)

6.6

34

24

22

(27)

6.4

17

24

19

(20)

3.6

 

150 µg

83

88

109

(93)

13.8

58

33

39

** (43)

13.1

34

40

34

(36)

3.5

13

27

34

(25)

10.7

13

10

15

(13)

2.5

 

500 µg

113

75

99

(96)

19.2

57

63

72

*** (64)

7.5

24

25

31

(27)

3.8

31

32

28

(30)

2.1

14

16

17

(16)

1.5

 

1500 µg

107 P

101 P

90 P

(99)

8.6

119 P

98 P

86 P

*** (101)

16.7

34 P

38 P

35 P

(36)

2.1

27 P

27 P

24 P

(26)

1.7

14 P

8 P

12 P

(11)

3.1

 

5000 µg

109 P

107 P

106 P

(107)

1.5

162 P

197 P

193 P

*** (184)

19.2

38 P

39 P

50 P

(42)

6.7

37 P

21 P

20 P

(26)

9.5

18 P

12 P

15 P

(15)

3.0

Positive controls S9-Mix (+)

 

Name DoseLevel

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2174

1392

2124

(1897)

437.8

282

276

299

(286)

11.9

228

328

323

(293)

56.3

213

233

251

(232)

19.0

511

497

526

(511)

14.5

BP         Benzo(a)pyrene

2AA    2-Aminoanthracene

P      Test itemprecipitate

**          p0.01

***        p0.001

#            Standarddeviation

Test Results: Confirmatory Experiment – With andWithoutMetabolic Activation(Pre-incubation)

 

 

Test Period

From: 19 October 2017

To: 22 October 2017

 

 

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strain

Without S9 TA1535

With S9 TA1535

Solvent Control (Acetone)

16

13

12

(14)

2.1#

12

10

12

(11)

1.2

 

15 µg

15

16

13

(15)

1.5

24

20

16

(20)

4.0

 

50 µg

12

25

23

(20)

7.0

43

24

38

*** (35)

9.8

 

150 µg

22

21

26

* (23)

2.6

51

56

80

*** (62)

15.5

 

500 µg

37

27

38

***  

(34)

6.1

80

69

67

*** (72)

7.0

 

1500 µg

64P

62P

73P

*** (66)

5.9

111P

94 P

95 P

*** (100)

9.5

 

5000 µg

99P

96P

77P

*** (91)

11.9

133P

133P

129P

*** (132)

2.3

 

Positive controls

 

Name DoseLevel

No. of Revertants

ENNG

2AA

5 µg

2 µg

417

501

561

(493)

72.3

321

315

347

(328)

17.0

 

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

2AA 2-Aminoanthracene

P                Test itemprecipitate

*                p 0.05

***           p0.001

#                Standard deviation


 

Test Results: Confirmatory Experiment – With andWithoutMetabolic Activation (PlateIncorporation)

 

Test Period

From: 19 October 2017

To: 22 October 2017

 

 

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strain

Without S9 TA1535

With S9 TA1535

Solvent Control (Acetone)

19

12

20

(17)

4.4#

12

9

23

(15)

7.4

 

15 µg

16

9

11

(12)

3.6

27

32

26

* (28)

3.2

 

50 µg

30

34

37

* (34)

3.5

84

58

71

*** (71)

13.0

 

150 µg

25

43

54

** (41)

14.6

120

103

108

*** (110)

8.7

 

500 µg

53

72

66

***(64)

9.7

173

156

155

*** (161)

10.1

 

1500 µg

113P

127P

107P

*** (116)

10.3

202P

180P

219P

*** (200)

19.6

 

5000 µg

198P

175P

237P

*** (203)

31.3

292P

345P

313P

*** (317)

26.7

 

Positive controls

 

Name DoseLevel

No. of Revertants

ENNG

2AA

5 µg

2 µg

345

383

420

(383)

37.5

191

146

173

(170)

22.6

                     


ENNG      N-ethyl-N'-nitro-N-nitrosoguanidine2AA2-Aminoanthracene

P                Test itemprecipitate

*                p0.05

**             p0.01

***           p0.001

#                Standard deviation

Applicant's summary and conclusion

Conclusions:
Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin was considered to be mutagenic under the conditions of this test only in S. typhimurium strain TA1535, this response was reproducible when utilizing the pre-incubation method.
Executive summary:

 Introduction

 The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

Methods

 Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods 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 (plate incorporation) was predetermined and was 1.5 to 5000mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. An additional dose level was selected in Experiment 2 in order to achieve both four non-toxic dose levels due to the change in methodology.

 

A Confirmatory Experiment was also performed in TA1535 (with and without metabolic activation) using the pre-incubation and plate incorporation methodology following the result from Experiment 2. The dose range was the same as Experiment 2 (15 to 5000 µg/plate).

 

 Results

 The vehicle (acetone) 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. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No toxicity was observed in the Confirmatory Experiment at any dose level, either in the presence or absence of metabolicactivation (S9-mix).

A greasy test item precipitate was noted at and above 1500mg/plate, this observation did not prevent the scoring of revertant colonies.


The test item induced statistically significant and reproducible increases in the frequency of TA1535 revertant colonies both with and without metabolic activation (S9-mix) in Experiment 2 and the Confirmatory Experiment (pre-incubation method). Statistically significant increases were also noted in the Confirmatory Experiment employing the plate incorporation method.

No significant increases in the frequency of revertant colonies were recorded in Experiment 1 or any of the remaining bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2.

 

Conclusion

Fatty acids, C18-unsatd., dimers, polymers with epichlorohydrin was considered to be mutagenic under the conditions of this test only inS. typhimuriumstrain TA1535, this response was reproducible when utilizing the pre-incubation method.