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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:
22 May - 11 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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
Specific details on test material used for the study:
Batch No.: 0400
Appearance: Solid, crystalline, deep orange powder
Storage conditions: Room temperature
Active components: NLT 96%

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 liver microsomal fraction
Test concentrations with justification for top dose:
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

The concentrations, including the controls were tested in triplicate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent waas compatible with the survival of the bacteria and the S9 activity.

The test item was dissolved in DMSO, processed by ultrasound for 5 min at 37°C and diluted prior to treatment.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD): TA98 and TA1537 (without S9) - 10 µg/plate for TA98 and 40 µg/ plate for TA1537; 2-aminoanthracene (2-AA): TA98, TA100, TA1535, TA1537 and TA102 (with S9) - 2.5 µg/plate and 10 µg/plate for TA102
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:
no 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 biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Balsalazide acid at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

HISTORICAL CONTROL DATA
- Positive historical control data: See Tables 4 and 6. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments. Only in experiment II, in tester strain TA 102 (with metabolic activation) a low mutation factor was found (1.9). Nevertheless, compared to the mutation factors found with the test item concentrations the increase can be considered as distinct. Moreover, the result is only slightly below the threshold value of 2.0. Thus, this effect was regarded as not biologically relevant.
- Negative (solvent/vehicle) historical control data: See Tables 3 and 5

INFORMATION ON CYTOTOXICITY:
- 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.

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

-

20

5.2

24

3.2

0.9

1.0

DMSO

-

22

1.5

24

3.0

1.0

1.0

Test item

31.6 µg

22

4.4

28

6.5

1.0

1.2

Test item

100 µg

19

3.5

23

1.5

0.9

1.0

Test item

316 µg

22

3.5

26

7.0

1.0

1.1

Test item

1000 µg

23

7.0

26

1.7

1.0

1.1

Test item

2500 µg

26

4.6

25

7.2

1.2

1.0

Test item

5000 µg

27

8.4

25

2.6

1.2

1.0

4-NOPD

10 µg

436

7.6

-

-

19.5

-

2-AA

2.5 µg

-

-

2145

145.8

-

89.4

TA100

Water

-

92

11.6

115

10.4

1.0

1.1

DMSO

-

88

8.4

107

14.0

1.0

1.0

Test item

31.6 µg

102

9.3

97

15.2

1.2

0.9

Test item

100 µg

104

12.9

114

9.8

1.2

1.1

Test item

316 µg

86

9.5

101

15.7

1.0

0.9

Test item

1000 µg

85

11.7

106

22.9

1.0

1.0

Test item

2500 µg

93

16.7

88

3.8

1.0

0.8

Test item

5000 µg

111

7.9

87

10.3

1.3

0.8

NaN3

10 µg

564

65.8

-

-

6.4

-

2-AA

2.5 µg

-

-

465

59.4

-

4.3

TA1535

Water

-

13

2.3

12

0.0

1.1

1.0

DMSO

-

11

1.2

12

3.5

1.0

1.0

Test item

31.6 µg

13

3.5

14

3.0

1.1

1.2

Test item

100 µg

13

1.0

13

3.1

1.1

1.1

Test item

316 µg

12

2.0

16

3.5

1.1

1.3

Test item

1000 µg

12

0.6

14

2.0

1.1

1.2

Test item

2500 µg

13

1.2

15

3.1

1.1

1.2

Test item

5000 µg

14

1.7

13

2.3

1.2

1.1

NaN3

10 µg

209

10.1

-

-

18.4

-

2-AA

2.5 µg

-

-

933

102.9

-

77.7

TA1537

Water

-

18

2.0

19

1.5

1.0

1.0

DMSO

-

19

2.1

20

2.1

1.0

1.0

Test item

31.6 µg

20

1.5

19

1.5

1.1

1.0

Test item

100 µg

19

2.1

19

1.7

1.0

0.9

Test item

316 µg

19

1.5

20

1.5

1.0

1.0

Test item

1000 µg

19

1.0

21

2.5

1.0

1.0

Test item

2500 µg

19

2.6

19

1.5

1.0

1.0

Test item

5000 µg

21

4.2

21

2.5

1.1

1.0

4-NOPD

40 µg

193

3.2

-

-

10.3

-

2-AA

2.5 µg

-

-

139

19.3

-

6.8

TA102

Water

-

314

44.0

258

46.0

1.0

1.0

DMSO

-

313

33.3

267

11.0

1.0

1.0

Test item

31.6 µg

319

27.5

257

18.5

1.0

1.0

Test item

100 µg

312

10.4

281

12.1

1.0

1.1

Test item

316 µg

315

7.4

275

31.5

1.0

1.0

Test item

1000 µg

324

24.6

215

34.5

1.0

0.8

Test item

2500 µg

311

7.8

231

21.4

1.0

0.9

Test item

5000 µg

324

16.0

185

10.0

1.0

0.7

MMS

1 µL

1148

94.2

-

-

3.7

-

2-AA

10 µg

-

-

1530

249.4

-

5.7

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

4.0

25

4.0

1.1

1.1

DMSO

-

22

5.9

23

5.3

1.0

1.0

Test item

31.6 µg

18

3.5

32

1.7

0.8

1.4

Test item

100 µg

26

8.6

27

3.5

1.1

1.2

Test item

316 µg

19

5.3

33

6.5

0.9

1.4

Test item

1000 µg

21

5.0

23

9.8

0.9

1.0

Test item

2500 µg

17

5.8

24

8.1

0.8

1.1

Test item

5000 µg

25

5.1

27

3.8

1.1

1.2

4-NOPD

10 µg

531

60.0

-

-

23.8

-

2-AA

2.5 µg

-

-

1125

7.0

-

48.9

TA100

Water

-

111

16.4

96

22.1

1.0

1.1

DMSO

-

107

16.9

90

21.6

1.0

1.0

Test item

31.6 µg

97

20.3

117

11.1

0.9

1.3

Test item

100 µg

126

21.6

103

9.6

1.2

1.2

Test item

316 µg

87

5.5

90

8.5

0.8

1.0

Test item

1000 µg

106

29.7

94

25.5

1.0

1.0

Test item

2500 µg

79

11.5

73

8.2

0.7

0.8

Test item

5000 µg

111

33.6

95

7.2

1.0

1.1

NaN3

10 µg

310

37.0

-

-

2.9

-

2-AA

2.5 µg

-

-

396

87.8

-

4.4

TA1535

Water

-

12

1.5

10

2.3

1.1

1.1

DMSO

-

11

1.5

10

2.5

1.0

1.0

Test item

31.6 µg

14

0.6

9

1.7

1.3

0.9

Test item

100 µg

12

2.1

12

3.5

1.1

1.2

Test item

316 µg

12

3.2

11

2.5

1.2

1.2

Test item

1000 µg

12

3.5

10

2.0

1.2

1.0

Test item

2500 µg

12

2.1

10

2.6

1.2

1.0

Test item

5000 µg

15

1.5

9

2.1

1.4

1.0

NaN3

10 µg

579

122.1

-

-

54.3

-

2-AA

2.5 µg

-

-

139

28.0

-

14.3

TA1537

Water

-

27

9.8

22

2.1

1.0

1.1

DMSO

-

26

7.2

20

3.2

1.0

1.0

Test item

31.6 µg

21

2.6

21

3.1

0.8

1.0

Test item

100 µg

24

0.6

21

2.6

0.9

1.0

Test item

316 µg

20

2.6

20

2.5

0.8

1.0

Test item

1000 µg

22

2.1

23

2.3

0.9

1.1

Test item

2500 µg

20

1.5

20

2.1

0.8

1.0

Test item

5000 µg

25

2.1

22

2.1

0.9

1.1

4-NOPD

40 µg

140

9.3

-

-

5.4

-

2-AA

2.5 µg

-

-

149

24.4

-

7.3

TA102

Water

-

268

22.0

295

20.7

1.0

0.9

DMSO

-

267

10.1

324

28.0

1.0

1.0

Test item

31.6 µg

276

9.1

306

20.8

1.0

0.9

Test item

100 µg

282

9.6

312

13.3

1.1

1.0

Test item

316 µg

251

6.6

285

12.8

0.9

0.9

Test item

1000 µg

284

9.1

287

15.0

1.1

0.9

Test item

2500 µg

248

15.7

278

21.1

0.9

0.9

Test item

5000 µg

263

24.3

278

25.4

1.0

0.9

MMS

1 µL

815

177.8

-

-

3.1

-

2-AA

10 µg

-

-

630

16.8

-

1.9

 

Table 3: Historical laboratory control data of the negative control (in 2015 – 2017) without S9

 

TA98

TA100

TA1535

TA1537

TA102

Mean

25.5

94.1

16.7

10.5

283.3

SD

6.8

16.6

6.6

5.5

53.5

Min

11

49

4

3

142

Max

58

155

41

35

472

RSD (%)

26.5

17.6

39.4

51.9

18.9

n

1071

1258

1040

1035

819

 

Table 4: Historical laboratory control data of the positive control (in 2015 – 2017) without S9

 

TA98

TA100

TA1535

TA1537

TA102

Substance

Conc/plate

4-NOPD

10 µg

NaN3

10 µg

NaN3

10 µg

4-NOPD

40 µg

MMS

1 µL

Mean

440.0

650.2

875.3

107.3

1610.3

SD

162.5

219.1

289.3

37.3

546.3

Min

89

146

56

36

412

Max

1895

2493

1854

570

3437

RSD (%)

36.9

33.7

33.1

34.7

33.9

n

1077

1264

1049

1039

824

 

Table 5: Historical laboratory control data of the negative control (in 2015 – 2017) with S9

 

TA98

TA100

TA1535

TA1537

TA102

Mean

29.3

95.5

13.3

10.7

345.7

SD

7.0

14.6

5.4

5.2

69.3

Min

15

60

3

3

157

Max

59

155

38

36

586

RSD (%)

23.8

15.3

40.4

48.1

20.0

n

1064

1252

1033

1028

812

 

Table 6: Historical laboratory control data of the positive control (in 2015 – 2017) with S9

 

TA98

TA100

TA1535

TA1537

TA102

Substance

Conc/plate

2-AA

2.5 µg

2-AA

2.5 µg

2-AA

2.5 µg

2-AA

2.5 µg

2-AA

10 µg

Mean

1615.9

1533.5

167.7

221.6

841.2

SD

687.6

563.7

158.2

110.5

215.0

Min

70

169

23

23

310

Max

3287

3092

1954

888

3588

RSD (%)

42.5

36.8

94.3

49.8

25.6

n

1067

1254

1039

1031

815

Applicant's summary and conclusion

Conclusions:
During the mutagenicity test under the experimental conditions reported, Balsalazide acid did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Balsalazide acid is considered to be non-mutagenic in this bacterial reverse mutation assay.