(3E)-3-[[4-(2-carboxyethylcarbamoyl)phenyl]hydrazinylidene]-6-oxocyclohexa-1,4-diene-1-carboxylic acid

Endpoint summary

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

Reference

Reference 1

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 2

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

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 1^stand 1000 for the 2^ndculture).

 

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.

Reference 3

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

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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]	RCE^a[%]	RTG^b[%]	MF^c[mutants/ 10^6cells]	IMF^d[mutants/ 10^6cells]	GEF^eexceeded	Statistical Significant Increase^f	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 = [(CE_{dose group}/ CE_{of 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.

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.