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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:
July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
GLP compliance:
yes (incl. certificate)
Remarks:
(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation: liquid
- Density:1.138 g/mL
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 12 Feb 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours has been verified analytically.
- Solubility and stability of the test substance in the 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.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in DMSO. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before administration.

Method

Target gene:
his, trp
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:
phenobarbital and ß-naphthoflavone induced rat liver S9 fraction
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2500 and 5000 µg/plate
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. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 µL/plate might also be tested in repeat experiments for further clarification / substantiation.
No mutagenicity was observed in the standard plate test.
Vehicle / solvent:
- Vehicle 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.
Controls
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
other: 2-aminoanthracene
Remarks:
positive control without S9 mix: 4-nitro-o-phenylenediamine, 10 µg/plate dissolved in DMSO, TA 98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h in the dark at 37 °C

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6); clearing or diminution of the background lawn (= reduced his- or trp- background growth)
Evaluation criteria:
Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings in the 1st experiment.

Toxicity
Toxicity detected by a
- decrease in the number of revertants (≤ 0.6)
- 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 and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Solubility
Precipitation of the test material was recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification / substantiation.
Statistics:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment 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 historical negative control data range 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:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit 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 nor precipitates, but tested up to recommended limit 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 nor precipitates, but tested up to recommended limit 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 nor precipitates, but tested up to recommended limit concentrations
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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 with and without S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: are given in the other information on material and methods
- Negative (solvent/vehicle) historical control data: are given in the other information on material and methods

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity: No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest required concentration.

Any other information on results incl. tables

Table 1: Standard Plate Test, without metabolic activation

Strain

Test group

Dose [µg/plate]

Mean revertants per plate

Standard deviation

Factor

Individual revertant colony counts

TA 1535

DMSO

Test item

 

 

 

 

 

MNNG

-

33

100

333

1000

2500

5000

5.0

7.0

7.7

11.3

7.0

11.3

10.0

12.0

4851.3

3.6

3.5

5.9

1.0

4.5

2.6

2.6

188.6

-

1.1

1.6

1.0

1.6

1.4

1.7

693.0

4, 11, 6

4, 11, 8

7, 18, 9

8, 6, 7

16, 7, 11

13, 8, 9

10, 15, 11

5069, 4749, 4736

TA 100

DMSO

Test item

 

 

 

 

 

MNNG

-

33

100

333

1000

2500

5000

5.0

102.7

100.0

92.0

102.3

106.7

94.3

93.0

3433.3

16.6

7.5

8.7

11.2

12.3

8.5

5.6

186.4

-

1.0

0.9

1.0

1.0

0.9

0.9

33.4

118, 85, 105

99, 108, 93

82, 98, 96

115, 98, 94

117, 93, 110

91, 104, 88

99, 92, 88

3605, 3460, 3235

TA 1537

DMSO

Test item

 

 

 

 

 

AAC

-

33

100

333

1000

2500

5000

100

8.3

5.0

9.0

6.7

11.3

8.7

8.3

1634.0

2.5

1.7

1.0

0.6

3.5

4.0

1.5

463.7

-

0.6

1.1

0.8

1.4

1.0

1.0

196.1

8, 6, 11

3, 6, 6

8, 10, 9

7, 7, 6

8, 11, 15

11, 11, 4

10, 8, 7

1649, 1163, 2090

TA 98

DMSO

Test item

 

 

 

 

 

NOPD

-

33

100

333

1000

2500

5000

10

19.0

16.7

15.0

13.7

19.7

17.3

16.0

419.7

2.0

1.5

1.7

3.8

6.7

1.5

3.5

41.8

-

0.9

0.8

0.7

1.0

0.9

0.8

22.1

17, 19, 21

18, 15, 17

13, 16, 16

12, 18, 11

24, 12, 23

19, 16, 17

12, 18, 18

437, 372, 450

E.coli

DMSO

Test item

 

 

 

 

 

4-NQO

-

33

100

333

1000

2500

5000

5

23.7

23.3

28.3

22.3

25.7

19.7

22.7

985.0

2.1

3.5

6.8

3.1

7.4

1.2

3.8

82.4

-

1.0

1.2

0.9

1.1

0.8

1.0

41.6

26, 22, 23

27, 23, 20

26, 36, 23

23, 19, 25

20, 34, 23

19, 19, 21

27, 20, 21

1080, 933, 942

Table 2: Standard Plate Test, with metabolic activation

Strain

Test group

Dose [µg/plate]

Mean revertants per plate

Standard deviation

Factor

Individual revertant colony counts

TA 1535

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

2.5

9.3

13.0

9.0

8.0

10.7

13.7

7.7

275.3

4.2

3.5

1.7

0.0

3.5

2.5

2.9

48.0

-

1.4

1.0

0.9

1.1

1.5

0.8

29.5

6, 14, 8

17, 11, 11

10, 7, 10

8, 8, 8

14, 11, 7

16, 11, 14

6, 6, 11

323, 227, 276

TA 100

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

2.5

106.0

104.0

96.3

93.0

98.7

100.7

98.0

1990.7

7.8

6.6

17.0

12.5

6.1

3.5

13.0

255.7

-

1.0

0.9

0.9

0.9

0.9

0.9

18.8

111, 110, 97

103, 98, 111

77, 109, 103

105, 80, 94

104, 92, 100

101, 97, 104

113, 91, 90

1948, 2265, 1759

TA 1537

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

2.5

6.7

9.3

6.3

8.3

9.0

10.7

9.3

136.0

1.2

1.2

1.2

1.5

6.2

3.1

2.5

20.8

-

1.4

1.0

1.3

1.4

1.6

1.4

20.4

8, 6, 6

10, 10, 8

7, 5, 7

7, 8, 10

11, 2, 14

10, 14, 8

7, 12, 9

148, 148, 112

TA 98

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

2.5

22.3

21.7

26.7

27.7

28.7

21.3

27.3

1558.7

4.5

2.5

8.7

3.8

2.9

2.5

10.5

265.0

-

1.0

1.2

1.2

1.3

1.0

1.2

69.8

18, 22, 27

22, 19, 24

17, 29, 34

25, 32, 26

27, 27, 32

21, 24, 19

17, 38, 27

1372, 1862, 1442

E.coli

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

60

23.0

29.0

27.0

21.0

23.7

32.7

20.7

83.0

6.1

2.6

3.6

7.9

5.5

2.3

2.5

5.0

-

1.3

1.2

0.9

1.0

1.4

0.9

3.6

19, 30, 20

27, 32, 28

24, 31, 26

18, 15, 30

21, 20, 30

34, 30, 34

21, 18, 23

88, 83, 78

Table 3: Preincubation Test, without metabolic activation

Strain

Test group

Dose [µg/plate]

Mean revertants per plate

Standard deviation

Factor

Individual revertant colony counts

TA 1535

DMSO

Test item

 

 

 

 

 

MNNG

-

33

100

333

1000

2500

5000

5.0

8.7

9.0

8.0

9.3

8.7

6.3

9.7

2417.3

1.2

0.0

2.0

2.5

3.1

2.1

4.5

175.3

-

1.0

0.9

1.1

1.0

0.7

1.1

278.9

10, 8, 8

9, 9, 9

10, 8, 6

7, 9, 12

12, 6, 8

8, 7, 4

5, 14, 10

2579, 2231, 2442

TA 100

DMSO

Test item

 

 

 

 

 

MNNG

-

33

100

333

1000

2500

5000

5.0

83.0

92.3

88.0

82.7

82.7

83.0

92.7

1636.7

3.5

4.6

17.7

4.2

7.2

2.0

16.3

122.0

-

1.1

1.1

1.0

1.0

1.0

1.1

19.7

87, 81, 81

95, 95, 87

107, 85, 72

84, 86, 78

91, 78, 79

81, 85, 83

100, 74, 104

1752, 1649, 1509

TA 1537

DMSO

Test item

 

 

 

 

 

AAC

-

33

100

333

1000

2500

5000

100

6.3

7.0

6.3

6.3

6.3

4.7

6.3

675.3

2.5

1.7

1.5

2.1

4.9

1.5

4.0

57.8

-

1.1

1.0

1.0

1.0

0.7

1.0

106.6

4, 6, 9

6, 6, 9

5, 6, 8

4, 8, 7

4, 3, 12

5, 6, 3

4, 4, 11

624, 664, 738

TA 98

DMSO

Test item

 

 

 

 

 

NOPD

-

33

100

333

1000

2500

5000

10

16.3

11.3

9.0

16.3

11.0

12.0

13.0

349.7

1.5

4.2

1.7

3.5

0.0

4.0

6.2

13.3

-

0.7

0.6

1.0

0.7

0.7

0.8

21.4

18, 15, 16

16, 10, 8

8, 11, 8

20, 13, 16

11, 11, 11

8, 12, 16

6, 15, 18

353, 335, 361

E.coli

DMSO

Test item

 

 

 

 

 

4-NQO

-

33

100

333

1000

2500

5000

5

21.0

22.0

11.0

21.0

21.3

17.3

17.3

453.3

2.0

7.5

1.0

1.0

1.5

4.9

4.2

57.1

-

1.0

0.5

1.0

1.0

0.8

0.8

21.6

19, 23, 21

21, 15, 30

12, 11, 10

20, 22, 21

20, 23, 21

15, 14, 23

15, 14, 22

466, 503, 391

Table 4: Preincubation Test, with metabolic activation

Strain

Test group

Dose [µg/plate]

Mean revertants per plate

Standard deviation

Factor

Individual revertant colony counts

TA 1535

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

2.5

12.3

10.7

11.3

11.7

8.0

8.3

10.7

194.0

3.2

4.7

3.1

2.5

4.0

1.5

3.5

6.6

-

0.9

0.9

0.9

0.6

0.7

0.9

15.7

11, 10, 16

9, 7, 16

12, 8, 14

12, 9, 14

4, 8, 12

7, 8, 10

14, 11, 7

195, 200, 187

TA 100

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

2.5

83.0

88.7

87.3

93.0

90.7

87.7

84.7

1550.7

8.5

13.6

12.5

15.4

8.3

15.3

4.2

79.7

-

1.1

1.1

1.1

1.1

1.1

1.0

18.7

92, 75, 82

87, 76, 103

96, 73, 93

89, 80, 110

88, 100, 84

97, 96, 70

80, 86, 88

1603, 1459, 1590

TA 1537

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

2.5

8.3

8.3

8.3

8.7

8.7

11.0

7.3

106.7

3.2

2.1

2.1

3.8

3.1

1.7

2.3

16.1

-

1.0

1.0

1.0

1.0

1.3

0.9

12.8

7, 12, 6

9, 10, 6

10, 9, 6

6, 13, 7

8, 12, 6

10, 13, 10

6, 6, 10

125, 95, 100

TA 98

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

2.5

19.0

24.0

18.0

24.3

17.3

12.7

20.0

508.7

3.6

2.0

4.6

4.7

6.5

3.5

1.0

12.9

-

1.3

0.9

1.3

0.9

0.7

1.1

26.8

15, 22, 20

26, 24, 22

19, 13, 22

28, 19, 26

17, 11, 24

13, 16, 9

21, 19, 20

518, 514, 494

E.coli

DMSO

Test item

 

 

 

 

 

2-AA

-

33

100

333

1000

2500

5000

60

19.0

21.0

14.0

18.7

22.0

15.3

20.0

69.0

1.0

4.6

1.0

2.1

6.6

6.4

2.6

18.5

-

1.1

0.7

1.0

1.2

0.8

1.1

3.6

19, 18, 20

26, 17, 20

13, 15, 14

17, 21, 18

29, 21, 16

8, 18, 20

22, 17, 21

87, 50, 70

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonelly typhimurium and Escherichia coli, in a reverse mutation assay.

Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Dose Range: 33 µg - 5000 µg/plate (SPT & PIT)

Test Conditions: Standard Plate Test (SPT) and Preincubation Test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

Solubility: No precipitation of the test substance was found with and without S9 mix.

Toxicity: No bacteriotoxic effect was observed under all test conditions.

Mutagenicity: A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (SPT and PIT assay).

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.