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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:
18 June 2018 - 28 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: B1187
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (under light exclusion)
- Stability under test conditions: The stability of the test substance under storage conditions is guaranteed by the sponsor.

Method

Target gene:
Salmonella typhimurium: The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined.
Escherichia coli: The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.
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:
S9 Mix from Wistar rat liver (phenobarbital and β-naphthoflavone induced)
Test concentrations with justification for top dose:
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment.
1st Experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (Standard plate test with and without S9 mix, TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA)
2nd Experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (Standard plate test, TA 1537 with and without S9 mix); 0; 0.33; 1.0; 3.3; 10; 33 and 100 μg/plate (Standard plate test, TA 100 and TA 98 both with S9 mix)

Reason for 2nd Experiment: Increase of revertants was observed in the standard plate test using the tester strains TA 100 and TA 98 with S9 mix. The Increase was observed from the 1st tested concentration onward, therefore the experiment was confirmed with adjusted dose levels. Furthermore, due to technical reason, the experiment with TA 1537 was not performed, therefore the part was repeated in the 2nd Experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
2.5 μg/plate (TA 1535, TA 100, TA 1537, TA 98), 60 μg/plate (Escherichia coli WP2 uvrA)
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
100 μg/plate (TA 1537)
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
5 μg/plate (E. coli WP2 uvrA)
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
5 μg/plate (TA 1535, TA 100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD)
Remarks:
10 μg/plate (TA 98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity detected by a decrease in the number of revertants (factor ≤ 0.6) and clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A weak bacteriotoxic effect (slight decrease in the number of trp+ revertants) was observed in the standard plate test at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A weak bacteriotoxic effect (slight decrease in the number of trp+ revertants) was observed in the standard plate test at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data: see table 1
- Positive historical control data: see table 2

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies compatible with the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at 5000 μg/plate.

Any other information on results incl. tables

Table 1: Historical Negative Controls (Feb 2016 - Feb 2017)

 Strain  S9 mix  Vehicle  No. of Plates  No. of Values  Min  Max  Mean  SD
 TA 1535 Without  (All)  414 152  7  16  10  2.0
  With  (All)  414 152   6  18  10  2.0
 TA 100 Without  (All)  426 152  71  132  100  11.4
  With   (All)  417  153  70  147  107  13.7
 TA 1537 Without  (All)  411  152  5  13  8  1.7
  With  (All)  411  152  5 16   9  2.1
 TA 98 Without  (All)  420  152  14 34   21  3.3
  With  (All)  413  152  12  38  28  4.5
 E.coli Without  (All)  396  152  15  34  24  3.9
  With  (All)  399  152  17  36  25 4.4 

Table 1: Historical Positive Controls (Feb 2016 - Feb 2017)

 Strain  S9 mix Positive control  No. of Plates  No. of Values  Min  Max  Mean  SD
 TA 1535 Without MNNG  312 110 1541  6171  3967  1253.9
  With  2 -AA  312 110  105  520  197  56.3
 TA 100 Without  MNNG  315 110  1126  5557  3298  1109.6
  With  2 -AA  309 111  272  3021  1748  587.2
 TA 1537 Without  AAC  309  110  253  2190 1044  404.3
  With 2 -AA  312  110  50 399 141  50.3
 TA 98 Without  NOPD  309  110  324 1746 863  206.8
  With 2 -AA  315  110  493  3096  1524  529.1
 E.coli Without 4 -NQO 306  110  164  1721  860  431.9
  With 2 -AA  306  110  61  537  133 61.8

Table 3: Without metabolic activation (Standard plate test)

Strain

 

 

 

 

Test group

 

 

 

 

Dose

(µg/plate)

 

 

 

Mean

revertants

per plate

 

Standard

deviation

 

 

 

Factor

 

 

 

 

Individual revertant colony

Counts

 

 

 

TA 1535

DMSO

-

10.0

1.7

-

9, 9, 12

 

Test item

33

8.0

3.5

0.8

4, 10, 10

 

 

100

11.0

1.7

1.1

10, 13, 10

 

 

333

11.0

5.3

1.1

15, 5,13

 

 

1000

8.3

2.5

0.8

8,6,11

 

 

2500

7.3

2.1

0.7

8,9,5

 

 

5000

8.7

1.2

0.9

10,8,8

 

MNNG

5.0

4972.7

63.7

497.3

4925,4948,5045

 

TA 100

DMSO

-

113.0

16.5

-

114,96,129

 

Test item

33

98.0

7.0

0.9

90, 103, 101

 

 

100

108.0

15.7

1.0

91,111,122

 

 

333

116.7

16.3

1.0

111,104,135

 

 

1000

138.3

9.5

1.2

148, 138, 129

 

 

2500

156.0

29.9

1.4

190,134,144

 

 

5000

127.3

3.2

1.1

125, 126, 131

 

MNNG

5.0

3406.0

612.2

30.1

3967,3498, 2753

 

TA 1537

DMSO

-

-

-

-

-T,-T,-T

 

Test item

33

-

-

-

- T, - T, - T

 

 

100

-

-

-

-T,-T,-T

 

 

333

-

-

-

-T,-T,-T

 

 

1000

-

-

-

-T,-T,-T

 

 

2500

-

-

-

-T,-T,-T

 

 

5000

-

-

-

-T,-T,-T

 

AAC

100

-

-

-

-T,-T,-T

 

TA 98

DMSO

-

19.0

5.3

-

15, 25, 17

 

Test item

33

24.3

5.9

1.3

31, 22, 20

 

 

100

18.3

1.2

1.0

17, 19, 19

 

 

333

18.7

5.1

1.0

13, 23, 20

 

 

1000

23.0

5.3

1.2

17, 27, 25

 

 

2500

22.7

7.6

1.2

14, 28, 26

 

 

5000

22.0

4.6

1.2

27, 18, 21

 

NOPD

10

774.7

51.1

40.8

725,772, 827

 

E. coli

DMSO

-

23.0

6.2

-

18, 21, 30

 

Test item

33

27.7

7.5

1.2

20, 28, 35

 

 

100

20.0

4.4

0.9

15, 23, 22

 

 

333

23.3

6.1

1.0

22, 18, 30

 

 

1000

22.7

4.7

1.0

19, 28, 21

 

 

2500

15.7

6.7

0.7

14, 23, 10

 

 

5000

9.3

2.1

0.4

11,7, 10

 

4-NQO

5

632.7

88.6

27.5

535,708, 655

T = Technical fault

Table 4: With metabolic activation (Standard plate test)

Strain

 

 

 

 

Test group

 

 

 

 

Dose

(µg/plate)

 

 

 

Mean

revertants

per plate

 

Standard

deviation

 

 

 

Factor

 

 

 

 

Individual revertant colony

Counts

 

 

 

TA 1535

DMSO

-

12.3

4.0

-

10,17,10

 

Test item

33

13.0

3.6

1.1

9,16,14

 

 

100

14.3

4.0

1.2

10,15,18

 

 

333

11.7

3.1

0.9

15,11,9

 

 

1000

14.0

3.6

1.1

15,17,10

 

 

2500

12.3

5.9

1.0

19,10,8

 

 

5000

7.0

1.0

0.6

7,6,8

 

2 -AA

2.5

172.3

13.1

14.0

187,168,162

 

TA 100

DMSO

-

110.0

6.9

-

114,114,102

 

Test item

33

354.0

33.2

3.2

389,350,323

 

 

100

525.7

40.4

4.8

549,479,549

 

 

333

746.3

65.9

6.8

674,803,762

 

 

1000

874.0

37.2

7.9

917,852,853

 

 

2500

804.3

72.9

7.3

856,836,721

 

 

5000

533.3

56.9

4.8

586,473,541

 

2 -AA

2.5

1269.0

249.5

11.5

1554,1090,1163

 

TA 1537

DMSO

-

-

-

-

-T,-T,-T

 

Test item

33

-

-

-

- T, - T, - T

 

 

100

-

-

-

-T,-T,-T

 

 

333

-

-

-

-T,-T,-T

 

 

1000

-

-

-

-T,-T,-T

 

 

2500

-

-

-

-T,-T,-T

 

 

5000

-

-

-

-T,-T,-T

 

2 -AA

2.5

-

-

-

-T,-T,-T

 

TA 98

DMSO

-

28.0

8.5

-

37,20,27

 

Test item

33

66.0

4.6

2.4

70,67,61

 

 

100

66.0

3.0

2.4

63,69,66

 

 

333

114.3

8.3

4.1

105,121,117

 

 

1000

119.3

4.0

4.3

123,120,115

 

 

2500

111.3

8.7

4.0

104,121,109

 

 

5000

80.7

17.0

2.9

98,80,64

 

2 -AA

2.5

1005.0

188.4

35.9

860,937,1218

 

E. coli

DMSO

-

30.0

7.8

-

39,25,26

 

Test item

33

28.3

4.5

0.9

28,24,33

 

 

100

28.0

8.2

0.9

21,37,26

 

 

333

39.3

3.1

1.3

36,40,42

 

 

1000

29.7

9.1

1.0

40,26,23

 

 

2500

20.3

9.3

0.7

16,14,31

 

 

5000

13.7

2.1

0.5

12,16,13

 

2 -AA

60

91.0

28.8

3.0

117,60,96

T = Technical fault

Table 5: Without metabolic activation (Standard plate test, 2nd Experiment)

 Strain

 Test group

 Dose (µg/mL)

 Mean revertants per plate

Standard devisation 

Factor 

 Individual revertant colony counts

 

DMSO

-

8.0

1.7

-

7, 10,7

 

Test item

33

11.3

5.5

1.4

14, 15,5

TA 1537

 

100

7. 3

2.5

0.9

10, 5,7

 

 

333

10.7

1.5

1.3

12, 9,11

 

 

1000

6.0

3.6

0.8

9,2,7

 

 

2500

6.0

2.6

0.8

8,3,7

 

 

5000

5.7

2.1

0.7

4, 5,8

 

AAC

100

875.0

287.1

109.4

726,693,1206

Table 5: With metabolic activation (Standard plate test, 2nd Experiment)

Strain

 

 

Test group

 

 

Dose

(µg/plate)

 

Mean

revertants
per plate

 

Standard

deviation

 

Factor

 

 

Individual revertant colony

counts

 

TA 100

DMSO

-

102.3

8.6

-

104,93, 110

 

Test item

0.33

91.7

3.2

0.9

88, 94,93

 

 

1.0

104.0

10.1

1.0

102, 115, 95

 

 

3.3

147.7

11.9

1.4

134,153, 156

 

 

10

187.7

18.9

1.8

181,173, 209

 

 

33

302.3

40.7

3.0

258,311, 338

 

 

100

466.3

93.0

4.6

374,465, 560

 

2-AA

2.5

1943.0

195.5

19.0

1756,1927, 2146

 

TA 1537

DMSO

-

8.3

1.2

-

9,7,9

 

Test item

33

8.7

3.2

1.0

10, 5, 11

 

 

100

9.3

6.0

1.1

10, 15, 3

 

 

333

6.0

3.6

0.7

10, 3, 5

 

 

1000

6.7

2.1

0.8

6, 5,9

 

 

2500

9.0

2.0

1.1

11,9,7

 

 

5000

7.7

4.0

0.9

4,7,12

 

2-AA

2.5

115.7

20.5

13.9

133, 93,121

 

TA 98

DMSO

-

19.0

1.7

-

20, 17, 20

 

Test item

0.33

23.3

2.5

1.2

23, 26, 21

 

 

1.0

22.0

4.0

1.2

26, 22, 18

 

 

3.3

29.0

5.0

1.5

34, 24, 29

 

 

'0

39.7

1.5

2.1

38, 40, 41

 

 

33

47.7

3.2

2.5

49, 44, 50

 

 

100

53.3

8.5

2.8

47, 63, 50

 

2-AA

2.5

1324.0

128.4

69.7

1390,1176, 1406

 

Applicant's summary and conclusion