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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:
30 April - 28 May 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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:
Batch: 12/18

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: MOLTOX, INC., NC 28607, USA (for TA98, TA1535 and TA102) and Xenometrix AG, Switzerland (for TA100 and TA1537)
Additional strain / cell type characteristics:
not applicable
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 / solvent:
- 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.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
A.dest
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without metabloic activation
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
plate incorporation test - EXPERIMENT I; pre-incubation test - EXPERIMENT II

For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
- 100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
- 2000 µL Overlay agar.

For the pre-incubation method 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: at least 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity can be detected by a clearing or 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 toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
Toxicity may 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.
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
Evaluation criteria:
A test item is considered 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 trater strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS: See Tables 1 and 2

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 4-Aminobenzoyl-b-alanine at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met

Any other information on results incl. tables

Table 1: Results Experiment I (plate incorporation test):

Treatment

Dose/plate

Revertant colonies per plate

Mutation factor

Without S9

With S9

Mean

SD

Mean

SD

-S9

+S9

TA98

Water

-

23

2.1

23

2.1

1.0

1.0

DMSO

-

24

2.5

24

1.5

1.0

1.0

Test item

31.6 µg

23

0.6

23

2.5

1.0

1.0

Test item

100 µg

23

2.0

23

1.0

1.0

1.0

Test item

316 µg

24

1.5

24

3.5

1.0

1.0

Test item

1000 µg

25

3.0

24

2.3

1.1

1.0

Test item

2500 µg

26

2.6

23

2.5

1.1

1.0

Test item

5000 µg

25

3.6

24

1.5

1.1

1.0

4-NOPD

10 µg

280

8.1

-

-

11.8

-

2-AA

2.5 µg

-

-

280

8.5

-

11.8

TA100

Water

-

82

1.5

82

3.6

1.0

1.0

DMSO

-

83

2.6

84

4.6

1.0

1.0

Test item

31.6 µg

84

4.6

84

2.5

1.0

1.0

Test item

100 µg

84

2.0

82

2.9

1.0

1.0

Test item

316 µg

83

3.1

85

2.6

1.0

1.0

Test item

1000 µg

84

2.5

83

5.2

1.0

1.0

Test item

2500 µg

86

1.7

84

10.0

1.0

1.0

Test item

5000 µg

89

2.1

85

6.2

1.1

1.0

NaN3

10 µg

457

64.4

-

-

5.5

-

2-AA

2.5 µg

-

-

480

61.5

-

5.7

TA1535

Water

-

21

1.7

21

3.1

1.0

1.0

DMSO

-

21

2.1

20

0.6

1.0

1.0

Test item

31.6 µg

21

2.0

21

3.1

1.0

1.0

Test item

100 µg

21

1.0

21

2.0

1.0

1.0

Test item

316 µg

23

4.4

21

1.5

1.1

1.0

Test item

1000 µg

23

2.5

22

1.2

1.1

1.1

Test item

2500 µg

23

2.1

23

2.9

1.1

1.1

Test item

5000 µg

24

3.8

23

4.2

1.1

1.1

NaN3

10 µg

272

24.5

-

-

12.8

-

2-AA

2.5 µg

-

-

246

14.5

-

12.1

TA1537

Water

-

20

2.9

20

4.2

1.0

1.0

DMSO

-

21

3.5

20

1.5

1.0

1.0

Test item

31.6 µg

21

2.5

21

3.5

1.0

1.0

Test item

100 µg

20

3.5

23

4.5

1.0

1.1

Test item

316 µg

20

2.5

22

5.0

1.0

1.1

Test item

1000 µg

21

3.5

23

2.3

1.0

1.1

Test item

2500 µg

21

2.5

24

4.5

1.0

1.2

Test item

5000 µg

22

3.5

25

3.8

1.0

1.2

4-NOPD

40 µg

144

12.9

-

-

6.8

-

2-AA

2.5 µg

-

-

226

21.7

-

11.1

TA102

Water

-

238

7.9

294

5.0

1.0

1.0

DMSO

-

240

8.6

298

6.2

1.0

1.0

Test item

31.6 µg

240

2.5

295

5.3

1.0

1.0

Test item

100 µg

240

4.0

297

5.7

1.0

1.0

Test item

316 µg

241

10.0

295

11.3

1.0

1.0

Test item

1000 µg

238

3.2

302

4.2

1.0

1.0

Test item

2500 µg

241

7.2

307

4.6

1.0

1.0

Test item

5000 µg

243

3.1

309

5.0

1.0

1.0

MMS

1 µL

1810

99.2

-

-

7.6

-

2-AA

10 µg

-

-

1455

379.3

-

4.9

 

Table 2: Results Experiment II (pre-incubation test):

Treatment

Dose/plate

Revertant colonies per plate

Mutation factor

Without S9

With S9

Mean

SD

Mean

SD

-S9

+S9

TA98

Water

-

24

1.0

24

2.3

1.0

1.0

DMSO

-

23

4.0

23

1.5

1.0

1.0

Test item

31.6 µg

23

2.5

23

3.1

1.0

1.0

Test item

100 µg

24

3.5

23

1.5

1.1

1.0

Test item

316 µg

22

2.6

25

2.5

1.0

1.1

Test item

1000 µg

24

1.5

25

1.5

1.0

1.1

Test item

2500 µg

25

2.0

23

1.2

1.1

1.0

Test item

5000 µg

24

1.0

25

4.6

1.0

1.1

4-NOPD

10 µg

225

13.7

-

-

9.8

-

2-AA

2.5 µg

-

-

208

95.6

-

9.2

TA100

Water

-

83

4.9

83

3.6

1.0

1.0

DMSO

-

82

5.7

81

10.7

1.0

1.0

Test item

31.6 µg

82

3.1

81

4.5

1.0

1.0

Test item

100 µg

82

4.4

81

6.7

1.0

1.0

Test item

316 µg

85

4.6

83

1.2

1.0

1.0

Test item

1000 µg

85

4.6

82

1.5

1.0

1.0

Test item

2500 µg

86

6.7

85

8.1

1.0

1.0

Test item

5000 µg

85

7.2

86

4.2

1.0

1.1

NaN3

10 µg

1302

68.6

-

-

15.8

-

2-AA

2.5 µg

-

-

343

20.8

-

4.2

TA1535

Water

-

22

2.1

20

2.0

1.0

0.9

DMSO

-

22

2.9

22

3.1

1.0

1.0

Test item

31.6 µg

21

1.7

22

1.7

0.9

1.0

Test item

100 µg

22

5.7

21

3.8

1.0

1.0

Test item

316 µg

21

1.2

22

3.8

0.9

1.0

Test item

1000 µg

23

2.6

20

1.5

1.0

0.9

Test item

2500 µg

24

3.2

23

5.5

1.1

1.0

Test item

5000 µg

24

4.0

23

5.6

1.1

1.1

NaN3

10 µg

245

7.9

-

-

11.0

-

2-AA

2.5 µg

-

-

249

17.7

-

11.5

TA1537

Water

-

20

2.9

20

0.6

1.0

1.0

DMSO

-

21

2.1

21

3.5

1.0

1.0

Test item

31.6 µg

21

1.0

21

5.0

1.0

1.0

Test item

100 µg

20

3.6

20

1.5

1.0

1.0

Test item

316 µg

21

2.5

22

1.2

1.0

1.1

Test item

1000 µg

21

4.2

23

4.7

1.0

1.1

Test item

2500 µg

22

2.0

22

4.4

1.1

1.1

Test item

5000 µg

23

1.2

22

1.5

1.1

1.1

4-NOPD

40 µg

203

8.7

-

-

9.8

-

2-AA

2.5 µg

-

-

179

11.5

-

8.6

TA102

Water

-

239

8.7

302

5.6

1.0

1.0

DMSO

-

241

1.0

301

6.0

1.0

1.0

Test item

31.6 µg

240

2.6

299

6.0

1.0

1.0

Test item

100 µg

242

9.6

301

2.5

1.0

1.0

Test item

316 µg

241

13.6

303

3.2

1.0

1.0

Test item

1000 µg

246

5.0

304

3.5

1.0

1.0

Test item

2500 µg

244

4.6

304

9.8

1.0

1.0

Test item

5000 µg

245

4.0

304

3.1

1.0

1.0

MMS

1 µL

1290

67.7

-

-

5.4

-

2-AA

10 µg

-

-

1188

48.6

-

3.9

 

Applicant's summary and conclusion

Conclusions:
4-Aminobenzoyl-b-alanine did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 4-Aminobenzoyl-b-alanine is considered to be non-mutagenic in this bacterial reverse mutation assay.