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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:
2016-10-0 to 2017-01-12
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st, July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30th, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
his
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-NOPD; 4-nitro-o-phenylene-diamine (without metabolic activation), 2-AA; 2-aminoanthracene (with metabolic activation)
Details on test system and conditions:
METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation) and Experiment II: preincubation
- Cell density at seeding (if applicable): 100 µL of Bacteria suspension containing approximately 1E09 cells/mL for both experimental settings

DURATION
- Preincubation period: 60 min in Experiment II
- Exposure duration: 48 h in both experimental settings

NUMBER OF REPLICATIONS: 3 replicates for each strain and dose level, including the controls


DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control.


Rationale for test conditions:
The OECD Guideline for Testing of Chemicals, Section 4, No. 471 — Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Results and discussion

Test results
Key result
Species / strain:
other: TA 98, TA, 100, TA 102, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations: 31.6, 100, 316, 1000,2500 and 5000 µg/plate.

Any other information on results incl. tables

Table 3:Number of revertants per plate (mean of 3 plates); incorporation method

 

[strain 1535]

[strain 1537]

[strain 102]

[strain 98]

[strain 100]

Conc.
[µg/plate]

— MA

+ MA

— MA

+ MA

— MA

+ MA

— MA

+ MA

— MA

+ MA

0 A dest.

29

15

5

6

270

370

19

28

115

117

0 *

28

14

11

12

261

339

27

28

120

110

31.6

29

17

11

10

282

383

18

22

114

113

100

22

12

11

7

275

342

18

17

128

127

316

24

11

10

9

268

307

21

24

116

129

1000

25

11

11

7

269

313

19

29

112

126

2500

25

15

9

9

319

308

23

24

98

112

5000

27

19

9

7

343

401

25

22

110

112

Positive control [-MA]

976

------

68

-------

1470

------

330

---------

757

-------

Positive control [+MA]

-------

276

-------

356

--------

925

-------

2405

------

2142

*solvent control with DMSO

Table 4:Number of revertants per plate (mean of 3 plates); preincubation method

 

[strain 1535]

[strain 1537]

[strain 102]

[strain 98]

[strain 100]

Conc.
[µg/plate]

— MA

+ MA

— MA

+ MA

— MA

+ MA

— MA

+ MA

— MA

+ MA

0 A dest.

7

16

6

9

236

303

19

22

85

108

0 *

8

12

7

7

206

316

22

17

73

95

31.6

5

14

5

9

214

295

21

14

69

93

100

8

13

5

6

219

358

20

20

85

93

316

5

13

4

5

247

307

24

19

91

110

1000

9

11

4

5

267

316

21

16

70

102

2500

3

16

6

7

252

305

21

25

84

96

5000

5

13

11

6

256

368

24

22

84

94

Positive control [-MA]

473

------

81

-------

1470

------

321

-------

573

-------

Positive control [+MA]

-------

164

-------

226

--------

677

-------

1751

------

1238

*solvent control with DMSO

Applicant's summary and conclusion

Conclusions:
The present study was conducted according to OECD Test Guideline 471 in order to determine the mutagenicity of L-asparagine. There was no increase in the amount of revertants for each incubation scheme and strain. Therefore, L-asparagine is considered to be non-mutagenic in the bacterial reverse mutation assay.