Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following OECD Guideline in vitro genotoxicity studies have been performed:

1). Bacterial reverse mutation test

2). In vitro mammalian cell gene mutation test – Mouse lymphoma assay

3). In vitro micronucleus test

The results of these tests were considered valid and all results were negative and no adverse effects were observed. It can therefore be concluded that balsalazide acid is not mutagenic.

Link to relevant study records

Referenceopen allclose all

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
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
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%
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.
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.
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.

 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

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.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 May - 13 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
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%
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ATCC, CCL-93
- Suitability of cells: These cells were chosen because of their stable karyotype and their low spontaneous induction rate of micronucleus formation under standardized culture conditions.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimum essential medium (MEM) supplemented with 10% FBS (fetal bovine serum)
- Properly maintained: yes - cells were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B (1.5 µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
The test item was prepared in cell culture medium within 1 hour prior to treatment After an ultrasonication treatment for 5 min at room temperature and vortexing a clear solution was obtained. The solvent was compatible with the survival of the cells and the S9 activity. The pH value measured was in the acidic range and was adjusted to the physiological range (7.0 ± 0.4) with 1 M NaOH.

Duplicate cultures were treated at each concentration. The following concentrations were used in the main experiments:
Experiment I: without and with metabolic activation: 125, 250, 500, 1000, 1500 and 2000 µg/mL
Experiment II: without metabolic activation: 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
other: Colchicine: Aneugenic control: 0.16 and 2.0 µg/mL (without S9)
Details on test system and experimental conditions:
EXPOSURE CONCENTRATIONS:
The following concentrations were used in the main experiments:
Experiment I: without and with metabolic activation: 125, 250, 500, 1000, 1500 and 2000 µg/mL
Experiment II: without metabolic activation: 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL

The following concentrations were selected for the microscopic analyses of micronuclei frequencies:
Experiment I with short-term exposure (4 h): without and with metabolic activation: 1000, 1500 and 2000 µg/mL
Experiment II with long-term exposure (24 h): without metabolic activation: 500, 1500 and 2000 µg/mL

EXPERIMENT I:
Exponentially growing V79 cells were seeded into 25 cm2 cell culture flasks (two flasks per test group). Approx. 50 000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 µg/mL cytochalasin B for 20 h at 37 °C.

EXPERIMENT II:
If negative or equivocal results were obtained, they were confirmed using continuous treatment (long-term treatment) without metabolic activation. Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm2 cell culture flasks in absence of metabolic activation. After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 µg/mL cytochalasin B were added and the cells were incubated for 23 h at 37 °C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.

PREPARATION OF THE CULTURES:
At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (ALSystems). After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.

METHOD OF APPLICATION: in medium
- Cell density at seeding: Approximately 50'000 cells were seeded per cell culture flask (25 cm2 flasks, two flasks per test group) containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS).

DURATION
- Exposure duration: Experiment I (with and without S9): 4 h; Experiment II (without S9): 24 h
- Cytochalasin B exposure duration: Experiment I: 20 h at 37°C; Experiment II: 23 h at 37°C
- Preparation interval: 24 h
- Total culture period (exposure started 48 h after culture initiation): 72 h

STAIN: Acridine orange solution

NUMBER OF CELLS EVALUATED: For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Cells were analysed for micronuclei according to the criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

DETERMINATION OF CYTOTOXICITY
- Method: As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells. The CBPI was then used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures.
Rationale for test conditions:
A pre-experiment was conducted under identical conditions as described for the main experiment I. The following concentrations were tested with and without S9 mix: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
The concentration of 2000 µg/mL was considered to be the highest test concentration used in this test system following the recommendation of the corresponding OECD testing guideline 487
Evaluation criteria:
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate of the test item was noted in any concentration evaluated in experiment I and II at the end of treatment with the unaided eye.

RANGE-FINDING/SCREENING STUDIES:
The test item was dissolved in MEM medium. No precipitate of the test item was noted at the end of treatment with the unaided eye. The highest dose group evaluated in the pre-experiment was 2000 µg/mL. The cytokinesis block proliferation index (CBPI) was used to calculate the cytostasis (cytostasis [%] = 100 - CBPI relative [%]). Cytostasis was used to describe cytotoxicity. The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment.

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: Not specified

NUMBER OF CELLS WITH MICRONUCLEI
In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.55%) was within the historical control limits of the negative control (0.37% – 1.37%). The mean values of micronucleated cells found after treatment with the test item were 0.75% (1000 µg/mL), 0.90% (1500 µg/mL) and 0.35% (2000 µg/mL). The numbers of micronucleated cells were within or below the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

In experiment I with metabolic activation the micronucleated cell frequency of the negative control (1.50%) was within the historical control limits of the negative control (0.42% – 1.64%). The mean values of micronucleated cells found after treatment with the test item were 1.60% (1000 µg/mL), 0.95% (1500 µg/mL) and 1.05% (2000 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

In experiment II without metabolic activation the micronucleated cell frequency of the negative control (0.55%) was within the historical control limits of the negative control (0.37% – 1.37%). The mean values of micronucleated cells found after treatment with the test item were 1.05% (500 µg/mL), 0.95% (1500 µg/mL) and 0.60% (2000 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

The nonparametric χ² Test was performed to verify the results in both experiments. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II with and without metabolic activation.

The χ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II (Table 8).

MMS (25 µg/mL) and CPA (5.0 µg/mL) were used as clastogenic controls and colchicine as aneugenic controls (0.16 and 2.0 µg/mL). They induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (2012 - 2017):
- MMS: Mean: 4.43 (Range 2.10 - 8.65); SD: 1.86; RSD: 41.93; LCL: 0.72; UCL: 8.15; n: 18
- Colchicine: Mean: 4.18 (Range 1.85 - 12.30); SD: 2.27; RSD: 54.30; LCL: 0.00; UCL: 8.72; n: 62
- CPA: Mean: 3.99 (Range 2.25 - 6.25); SD: 1.14; RSD: 28.54; LCL: 1.71; UCL: 6.27; n: 35
- Negative historical control data (2012 - 2017): Cell culture medium
- Without metabolic activation: Mean: 0.87 (Range 0.45 - 1.50); SD: 0.25; RSD: 28.75; LCL: 0.37; UCL: 1.37; n: 62
- With metabolic activation: Mean: 1.03 (Range 0.55 - 1.75); SD: 0.31; RSD: 29.76; LCL: 0.42; UCL: 1.64; n: 35
- Solvent historical control data (2012 - 2017): DMSO 1% v/v or ethanol 0.5% v/v in cell culture medium
- Without metabolic activation: Mean: 0.97 (Range 0.55 - 1.40); SD: 0.25; RSD: 25.87; LCL: 0.47; UCL: 1.48; n: 33
- With metabolic activation: Mean: 1.05 (Range 0.55 - 1.8); SD: 0.35; RSD: 33.49; LCL: 0.35; UCL: 1.75; n: 17

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
In experiment I with and without metabolic activation no increase of the cytostasis above 30% was noted.
In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 500 µg/mL. At 1500 µg/mL a cytostasis of 43% and at 2000 µg/mL a cytostasis of 55% was noted.

Table 1: Main Experiment I - CBPI: 4 h treatment (without and with metabolic activation), 24 h fixation period

Dose Group

Concentration

(µg/mL)

CBPI

1/2

CBPI

2/2

Relative cell growth

(%)

Cytostasis

(%)

Precipitate

+/-

Without metabolic activation

C

0

1.38

1.38

100

0

-

4

1000

1.37

1.40

101

0

-

5

1500

1.48

1.47

124

0

-

6

2000

1.48

1.46

124

0

-

MMS

25

1.30

1.31

80

20

-

Colchicine

2.0

1.46

1.46

120

0

-

With metabolic activation

C

0

1.43

1.49

100

0

-

4

1000

1.45

1.47

99

1

-

5

1500

1.52

1.55

116

0

-

6

2000

1.51

1.52

112

0

-

CPA

5.0

1.19

1.19

41

59

-

The CBPI was determined in 500 cells per culture of each test group.

The relative values of the CBPI are related to the negative control.

C:                        Negative Control (Culture medium)

MMS:                 Methylmethanesulfonate, Positive Control (without metabolic activation)

Colchicine:         Positive Control (without metabolic activation)

CPA:                    Cyclophosphamide, Positive Control (with metabolic activation)

CBPI:                  Cytokinesis Block Proliferation Index, CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3))/n

Relative Cell Growth:      100 x ((CBPI Test conc – 1) / (CBPI control -1))

c1: mononucleate cells

c2: binucleate cells

cx: multinucleate cells

n: total number of cells

CBPI ½:                            Slide 1 of 2

CBPI 2/2:            Slide 2 of 2

Cytostasis [%] = 100- Relative Cell Growth [%]; the cytostasis is defined 0, when the relative cell growth exceeds 100%

 

Table 2: Main Experiment I - Micronucleus induction in V79 cells, 4 h treatment, 24 h fixation interval, without metabolic activation

Dose group

Concentration

(µg/mL)

Scored binucleated cells

(total of 2 cultures)

Micronuclei

(total of 2 cultures)

Micronucleated cells frequency

(%, average)

C

0

2000

11

0.55

4

1000

2000

15

0.75

5

1500

2000

18

0.90

6

2000

2000

7

0.35

MMS

25

2000

60

3.00

Colchicine

2.0

1868

55

2.94

The micronucleated cell frequency was determined where possible in 1000 binucleated cells in each of the two separate cultures per test group, except for the positive control colchicine (868 for the 1stand 1000 for the 2ndculture).

 

Table 3: Main Experiment I - Micronucleus induction in V79 cells 4 h treatment, 24 h fixation interval, with metabolic activation

Dose group

Concentration

(µg/mL)

Scored binucleated cells

(total of 2 cultures)

Micronuclei

(total of 2 cultures)

Micronucleated cells frequency

(%, average)

C

0

2000

30

1.50

4

1000

2000

32

1.60

5

1500

2000

19

0.95

6

2000

2000

21

1.05

CPA

5.0

2000

180

9.00

 

Table 4: Main Experiment II - CBPI: 24 h treatment (without metabolic activation)

Dose Group

Concentration

(µg/mL)

CBPI

1/2

CBPI

2/2

Relative cell growth

(%)

Cytostasis

(%)

Precipitate

+/-

Without metabolic activation

C

0

1.62

1.54

100

0

-

4

500

1.41

1.44

74

26

-

6

1500

1.34

1.32

57

43

-

7

2000

1.25

1.27

45

55

-

MMS

25

1.43

1.45

76

24

-

Colchicine

0.16

1.34

1.24

51

49

-

 

Table 5: Main Experiment II - Micronucleus induction in V79 cells, 24 h treatment, without metabolic activation

Dose group

Concentration

(µg/mL)

Scored binucleated cells

(total of 2 cultures)

Micronuclei

(total of 2 cultures)

Micronucleated cells frequency

(%, average)

C

0

2000

11

0.55

4

500

2000

21

1.05

6

1500

2000

19

0.95

7

2000

2000

12

0.60

MMS

25

2000

94

4.70

Colchicine

0.16

1852

82

4.46

 

 

Table 6: Summary of Micronuclei Effects: Experiment I without and with metabolic activation

Dose group

Concentration

(µg/mL)

Treatment time

(h)

Fixation interval

(h)

Micronucleated Cells Frequency

(%)

Without metabolic activation

C

0

4

24

0.55

4

1000

4

24

0.75

5

1500

4

24

0.90

6

2000

4

24

0.35

MMS

25

4

24

3.00

Colchicine

2.0

4

24

2.94

With metabolic activation

C

0

4

24

1.50

4

1000

4

24

1.60

5

1500

4

24

0.95

6

2000

4

24

1.05

CPA

5.0

4

24

9.00

 

Table 7: Summary of Micronuclei Effects: Experiment II without metabolic activation

Dose group

Concentration

(µg/mL)

Treatment time

(h)

Fixation interval

(h)

Micronucleated Cells Frequency

(%)

Without metabolic activation

C

0

24

24

0.55

4

1000

24

24

1.05

6

1500

24

24

0.95

7

2000

24

24

0.60

MMS

25

24

24

4.70

Colchicine

0.16

24

24

4.46

 

Table 8: Biometry – Trend test

Statistical significance at the 5% level (p < 0.05) was evaluated by the χ² test for trend. The p value was used as a limit in judging for significance levels.

Experiment

Treatment time

(h)

Significance

P value

Exp. I without metabolic activation

4

-

0.1201

Exp. I with metabolic activation

4

-

0.1102

Exp. II without metabolic activation

24

-

0.1247

+:  significant

-: not significant

Statistical significance: statistical significant concentration-related increase in micronucleated cells frequency (χ² test for trend, p < 0.05)

Conclusions:
During the study described and under the experimental conditions reported, the test item Balsalazide acid did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, Balsalazide acid is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 September - 29 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
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%

PREPARATION OF THE TEST ITEM
Based on a pre-experiment for solubility, RPMI cell culture medium (+ 5% HS) was used as solvent and 2 mg/mL was used as the highest concentration.
The solvent was compatible with the survival of the cells and the S9 activity. The preparation of the test item did reveal changes in pH-value into a slight acid range. This change was adjusted with sodium hydroxide to achieve the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration was 309 mOsm/kg.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich.
- Suitability of cells: Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C) have been used successfully in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10 - 12 h doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The selection of the concentrations used in the main experiment was based on data from the pre-experiment. 2000 µg/mL (without and with metabolic activation) was selected as the highest concentration. The experiment without and with metabolic activation was performed as 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation:
25, 50, 100, 250, 500, 1000 and 2000 µg/mL

and with metabolic activation:
25, 50, 100, 250, 500, 1000 and 2000 µg/mL
Vehicle / solvent:
None
Untreated negative controls:
yes
Remarks:
Treatment medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
PRE-EXPERIMENT FOR TOXICITY
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 2 mg/mL. Six concentrations [50, 100, 250, 500, 1000 and 2000 µg/mL] were tested without and with metabolic activation. The experimental conditions in these pre-experiments were the same as for the definitive test. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated

EXPERIMENTAL PERFORMANCE
For a short-term exposure experiment 1x10^7 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h the test item was removed by centrifugation (200 x g, 7 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period of 2 days in total at 37 °C in 5% CO2/95% humidified air. The cell density was determined each day and adjusted to 3x10^5 cells/mL in a total culture volume of 20 mL, if necessary.

After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 7 days at 37 °C in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells) and those without cell growth (negative wells) compared to the total number of cultures seeded. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT. The plates were scored after an incubation period of about 12 days at 37 °C in 5% CO2/95% humidified air.

METHOD OF APPLICATION: suspended in medium
- Cell density: 1x10^7 cells suspended in 11 mL RPMI medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days at 37°C
- Selection time (if incubation with a selection agent): About 12 days at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): Triflurothymidine (TFT)

MUTATION FREQUENCY
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to –ln (negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per well). Mutant frequency then was calculated as MF = (PE(cultures in selective medium)/PE(cultures in non-selective medium)). The mutant frequency is usually expressed as “mutants per 10^6 viable cells”.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the microwell method.

SUSPENSION GROWTH
Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density. When carrying out a short-term treatment (4 h) a 2-day growth period was considered. The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100.
Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:

- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.

A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection.

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRECIPITATION
No precipitation of the test item was noted in the experiments.

TOXICITY
No growth inhibition was observed without and with metabolic activation.

MUTAGENICITY
In the main experiment without and with metabolic activation all validity criteria were met. The negative controls showed mutant frequencies within the acceptance range of 50 - 170 mutants/10^6 cells, according to the IWGT criteria.
No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor of 126 (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. None of the observed mutant frequencies was statistically significantly increased over those of the negative controls
No dose-response relationship was observed.
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

CLASTOGENICITY
Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) is an indication for potential clastogenic effects and/or chromosomal aberrations. Thus based on the non-mutagenic effects of Balsalazide acid, an assessment of clastogenicity was not feasible.
The positive controls MMS and B[a]P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects.

Table1:  Summary: Main Experiment, without and with metabolic activation

 

Test Group

Conc.

[µg/mL]

RCEa[%]

RTGb[%]

MFc[mutants/ 106cells]

IMFd[mutants/ 106cells]

GEFeexceeded

Statistical

Significant Increasef

Precipitate

Exp

without

S9

 

C1

0

100.0

100.0

94.2

/

/

/

-

C2

/

/

/

-

2

25

108.2

104.9

114.2

20.0

-

-

-

3

50

89.3

83.8

102.7

8.5

-

-

-

4

100

86.7

85.0

124.4

30.3

-

-

-

5

250

108.2

93.3

102.1

7.9

-

-

-

6

500

104.7

96.9

110.3

16.1

-

-

-

7

1000

118.2

84.0

68.0

-26.2

-

-

-

8

2000

96.5

79.1

145.7

51.5

-

-

-

EMS

300

81.7

71.0

661.7

567.5

+

+

-

MMS

10

61.3

44.7

819.0

724.8

+

+

-

 

Exp

with

S9

 

C1

0

100.0

100.0

114.7

/

/

/

-

C2

/

/

/

-

2

25

87.3

90.7

131.9

17.2

-

-

-

3

50

88.6

89.1

102.9

-11.8

-

-

-

4

100

101.6

105.6

110.0

-4.8

-

-

-

5

250

101.6

101.7

113.1

-1.6

-

-

-

6

500

94.0

104.9

122.2

7.5

-

-

-

7

1000

103.2

109.7

119.6

4.9

-

-

-

8

2000

91.3

97.9

120.6

5.9

-

 

-

B[a]P

2.5

87.3

38.1

710.4

595.7

+

+

-

C:      Negative Controls
a:
       Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]
           Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)
b:
      Relative Total Growth, RTG = (RSG x RCE)/100
c:
       Mutant Frequency,
           MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
d:
      Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
e:
       Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded
f:
       statistical significant increase in mutant frequency compared to negative controls (Mann Whitney test, p<0.05).
+: significant; -not significant
EMS:
  Ethylmethanesulfonate
MMS:Methylmethanesulfonate
B[a]P:Benzo[a]pyrene

Conclusions:
In conclusion, the test item Balsalazide acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The mode of action of toxicity and its relevance to humans has not been assessed. However, the sodium salt of balsalazide acid has been authorised for use as a pharmaceutical for the treatment of inflammatory bowel disease.

Additional information

Justification for classification or non-classification

Based on the negative results observed in three valid OECD Guideline in vitro genotoxicity studies, balsalazide acid is not classified as mutagenic or genotoxic.