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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:
October - November 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide
EC Number:
700-457-2
Molecular formula:
Unspecified
IUPAC Name:
Reaction mass of N-butylphosphorothioic triamide and N-propylphosphorothioic triamide
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 8712 / 056
- Purity: 99.6 g / 100 g
- Date of production: 01 Oct 2007
- Physical state and appearance: solid, white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator - 20 °C
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle water over a period of 4 hours was verified analytically.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in water. To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. 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:
Aroclor induced rat liver S9 fraction
Test concentrations with justification for top dose:
1st Experiment (Standard Plate Test): 0; 20; 100; 500; 2500 and 5000 µg/plate
2nd Experiment (Preincubation Test): 0; 312.5; 625; 1250; 2500 and 5000 µg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 µL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be 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 calculation / substantiation.
Vehicle / solvent:
- Vehicle used: water
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.
Controls
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
vehicle control
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: - 2-aminoanthracene (2-AA): with S9 mix, 2.5 µg/plate dissolved in DMSO (TA 1535, TA 100, TA 1537, TA 98) or 60 µg/plate dissolved in DMSO (E.coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: 48 - 72 hours in the dark at 37 °C

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer
Rationale for test conditions:
Bacterial reverse mutation assays using amino-acid requiring strains of Salmonella typhimurium and Escherichia coli are commonly employed as initial screening methods for detecting a point mutagenic activity of chemical substances and are used to screen for possible mammalian mutagens and carcinogens.
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, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is 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 are based on the findings of the 1st Experiment.
- Titer: The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
- Toxicity: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.
- Solubility: Precipitation of the test material is recorded and indicated in the tables. As long as precipitation does not interefere with the colony scoring, 5 mg/plate is 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 is considered valid if the following criteria are 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.
- The titer of viable bacteria was >= 10^8 / mL.

Assessment criteria
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in preincubation test only at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
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
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
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
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
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 found.

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

Any other information on results incl. tables

Table 1: Historical Negative Control Data TA 1535

Method

S9 mix

Negative control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

01.00 – 02.07

400

10

24

17

2

SPT

-

DMSO

11.04 – 02.07

400

10

25

17

2

SPT

-

Acetone

01.96 – 11.06

259

11

25

18

2

SPT

1 : 9

Water

01.00 – 01.07

400

12

24

18

2

SPT

1 : 9

DMSO

11.04 – 03.07

400

10

24

17

2

SPT

1 : 9

Acetone

01.96 – 11.06

258

11

25

19

2

PIT

-

Water

04.98 – 02.07

400

10

25

18

2

PIT

-

DMSO

07.03 – 03.07

400

10

25

17

2

PIT

-

Acetone

01.96 – 11.06

216

10

25

18

3

PIT

1 : 9

Water

11.97 – 02.07

400

11

25

18

2

PIT

1 : 9

DMSO

06.03 – 03.07

400

10

24

16

2

PIT

1 : 9

Acetone

01.96 – 11.06

201

10

24

18

2

*: revertants/plate

Table 2: Historical Negative Control Data TA 100

Method

S9 mix

Negative control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

12.99 – 02.07

400

82

150

110

9

SPT

-

DMSO

11.04 – 02.07

400

80

135

109

9

SPT

-

Acetone

01.96 – 11.06

306

91

160

117

14

SPT

1 : 9

Water

12.99 – 01.07

400

88

152

115

2

SPT

1 : 9

DMSO

11.04 – 03.07

400

81

154

111

11

SPT

1 : 9

Acetone

01.96 – 11.06

301

85

160

119

15

PIT

-

Water

04.98 – 02.07

400

90

160

115

13

PIT

-

DMSO

09.03 – 03.07

400

81

153

109

10

PIT

-

Acetone

01.96 – 11.06

223

91

160

116

14

PIT

1 : 9

Water

03.98 – 02.07

400

92

159

120

15

PIT

1 : 9

DMSO

07.03 – 03.07

400

81

157

110

10

PIT

1 : 9

Acetone

05.96 – 11.06

198

96

160

118

15

*: revertants/plate

Table 3: Historical Negative Control Data TA 1537

Method

S9 mix

Negative control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

11.99 – 02.07

400

5

16

10

2

SPT

-

DMSO

10.04 – 03.07

400

5

16

10

2

SPT

-

Acetone

01.96 – 11.06

261

5

18

10

2

SPT

1 : 9

Water

10.99 – 01.07

400

6

17

11

2

SPT

1 : 9

DMSO

09.04 – 03.07

400

5

16

10

2

SPT

1 : 9

Acetone

01.96 – 11.06

261

5

20

11

2

PIT

-

Water

04.98 – 02.07

400

5

16

10

2

PIT

-

DMSO

07.03 – 03.07

400

5

17

10

2

PIT

-

Acetone

01.96 – 11.06

213

6

17

10

2

PIT

1 : 9

Water

11.97 – 02.07

400

6

20

11

2

PIT

1 : 9

DMSO

07.03 – 03.07

400

5

17

10

2

PIT

1 : 9

Acetone

01.96 – 11.06

204

5

20

11

2

*: revertants/plate

Table 4: Historical Negative Control Data TA 98

Method

S9 mix

Negative control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

01.00 – 02.07

400

17

44

29

5

SPT

-

DMSO

10.04 – 03.07

400

15

40

29

4

SPT

-

Acetone

01.96 – 11.06

263

19

50

29

5

SPT

1 : 9

Water

01.00 – 01.07

400

20

49

36

5

SPT

1 : 9

DMSO

10.04 – 03.07

400

21

50

35

5

SPT

1 : 9

Acetone

01.96 – 11.06

262

25

50

38

5

PIT

-

Water

01.98 – 02.07

400

18

44

28

4

PIT

-

DMSO

07.03 – 03.07

400

17

40

29

4

PIT

-

Acetone

01.96 – 11.06

216

19

45

28

5

PIT

1 : 9

Water

11.97 – 02.07

400

17

49

35

2

PIT

1 : 9

DMSO

07.03 – 03.07

400

20

50

33

5

PIT

1 : 9

Acetone

01.96 – 11.06

203

20

50

36

6

*: revertants/plate

Table 5: Historical Negative Control Data E.coli WP2 uvrA

Method

S9 mix

Negative control

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

Water

09.99 – 01.07

400

25

55

33

5

SPT

-

DMSO

09.04 – 03.07

400

25

59

34

5

SPT

-

Acetone

04.96 – 11.06

234

25

55

34

5

SPT

1 : 9

Water

09.99 – 01.07

400

25

56

36

6

SPT

1 : 9

DMSO

09.04 – 03.07

400

25

60

38

6

SPT

1 : 9

Acetone

04.96 – 11.06

242

25

59

37

6

PIT

-

Water

10.97 – 02.07

400

25

52

32

5

PIT

-

DMSO

06.03 – 03.07

400

25

54

33

5

PIT

-

Acetone

01.96 – 11.06

191

25

54

32

5

PIT

1 : 9

Water

11.97 – 02.07

400

25

57

37

6

PIT

1 : 9

DMSO

07.03 – 03.07

400

25

56

36

5

PIT

1 : 9

Acetone

01.96 – 11.06

197

25

52

36

6

*: revertants/plate

Table 6: Historical Positive Control Data TA 1535

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

MNNG 5.0

03.05 – 02.07

400

509

1602

752

187

SPT

1 : 9

2-AA 2.5

02.05 – 03.07

400

77

342

132

28

PIT

-

MNNG 5.0

06.04 – 03.07

400

512

1317

708

150

PIT

1 : 9

2-AA 2.5

03.04 – 03.07

400

83

398

130

33

*: revertants/plate

Table 7: Historical Positive Control Data TA 100

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

MNNG 5.0

02.05 – 03.07

400

500

1574

823

201

SPT

1 : 9

2-AA 2.5

02.05 – 03.07

400

521

1878

876

205

PIT

-

MNNG 5.0

06.04 – 03.07

400

520

1302

797

141

PIT

1 : 9

2-AA 2.5

04.04 – 03.07

400

517

1497

829

174

*: revertants/plate

Table 8: Historical Positive Control Data TA 1537

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

AAC 100

02.05 – 03.07

400

212

1172

441

109

SPT

1 : 9

2-AA 2.5

02.05 – 03.07

400

72

200

129

22

PIT

-

AAC 100

05.04 – 03.07

400

221

1028

429

100

PIT

1 : 9

2-AA 2.5

03.04 – 03.07

400

80

198

123

19

*: revertants/plate

Table 9: Historical Positive Control Data TA 98

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

NOPD 10

02.05 – 03.07

400

332

1316

616

145

SPT

1 : 9

2-AA 2.5

02.05 – 03.07

400

502

1414

697

160

PIT

-

NOPD 10

06.04 – 03.07

400

388

1430

630

169

PIT

1 : 9

2-AA 2.5

03.04 – 03.07

400

510

1223

663

120

*: revertants/plate

Table 10: Historical Positive Control Data E.coli WP2 uvrA

Method

S9 mix

Positive control µg/plate

Period

No. of plates

Min*

Max*

Mean*

SD

SPT

-

4-NQO 5.0

01.05 – 03.07

400

500

1587

653

188

SPT

1 : 9

2-AA 60.0

01.05 – 03.07

400

150

383

231

30

PIT

-

4-NQO 5.0

05.04 – 03.07

400

500

1339

593

102

PIT

1 : 9

2-AA 60.0

05.04 – 03.07

400

150

307

226

25

*: revertants/plate

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. Salmonella 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: 20 µg - 5000 µg/plate (SPT), 312.5 µg - 5000 µg/plate (PIT)

Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).

Solubility: No precipitation of the test substance was found.

Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.

Mutagenicity: A 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 is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay under the experimental conditions chosen here.