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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May - 11 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

617-116-8

Cas Number:

80573-04-2

Molecular formula:

C17 H15 N3 O6

IUPAC Name:

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

Test material form:

solid

Specific details on test material used for the study:

Batch No.: 0400
Appearance: Solid, crystalline, deep orange powder
Storage conditions: Room temperature
Active components: NLT 96%

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MOLTOX, INC., NC 28607, USA (for TA98, TA1535 and TA102) and Xenometrix AG, Switzerland (for TA100 and TA1537)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

The concentrations, including the controls were tested in triplicate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent waas compatible with the survival of the bacteria and the S9 activity.

The test item was dissolved in DMSO, processed by ultrasound for 5 min at 37°C and diluted prior to treatment.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD): TA98 and TA1537 (without S9) - 10 µg/plate for TA98 and 40 µg/ plate for TA1537; 2-aminoanthracene (2-AA): TA98, TA100, TA1535, TA1537 and TA102 (with S9) - 2.5 µg/plate and 10 µg/plate for TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test - EXPERIMENT I; pre-incubation test - EXPERIMENT II

For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
- 100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
- 2000 µL Overlay agar.
For the pre-incubation method 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates (were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

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

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

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

Rationale for test conditions:

The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Evaluation criteria:

A test item is considered mutagenic if:
- a clear and dose related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one trater strain with or without metabolic activation.

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS: See Tables 1 and 2
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Balsalazide acid at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

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

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

INFORMATION ON CYTOTOXICITY:
- No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.

Any other information on results incl. tables

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

Treatment	Dose/plate	Revertant colonies per plate				Mutation factor
		Without S9		With S9
		Mean	SD	Mean	SD	-S9	+S9
TA98
Water	-	20	5.2	24	3.2	0.9	1.0
DMSO	-	22	1.5	24	3.0	1.0	1.0
Test item	31.6 µg	22	4.4	28	6.5	1.0	1.2
Test item	100 µg	19	3.5	23	1.5	0.9	1.0
Test item	316 µg	22	3.5	26	7.0	1.0	1.1
Test item	1000 µg	23	7.0	26	1.7	1.0	1.1
Test item	2500 µg	26	4.6	25	7.2	1.2	1.0
Test item	5000 µg	27	8.4	25	2.6	1.2	1.0
4-NOPD	10 µg	436	7.6	-	-	19.5	-
2-AA	2.5 µg	-	-	2145	145.8	-	89.4
TA100
Water	-	92	11.6	115	10.4	1.0	1.1
DMSO	-	88	8.4	107	14.0	1.0	1.0
Test item	31.6 µg	102	9.3	97	15.2	1.2	0.9
Test item	100 µg	104	12.9	114	9.8	1.2	1.1
Test item	316 µg	86	9.5	101	15.7	1.0	0.9
Test item	1000 µg	85	11.7	106	22.9	1.0	1.0
Test item	2500 µg	93	16.7	88	3.8	1.0	0.8
Test item	5000 µg	111	7.9	87	10.3	1.3	0.8
NaN3	10 µg	564	65.8	-	-	6.4	-
2-AA	2.5 µg	-	-	465	59.4	-	4.3
TA1535
Water	-	13	2.3	12	0.0	1.1	1.0
DMSO	-	11	1.2	12	3.5	1.0	1.0
Test item	31.6 µg	13	3.5	14	3.0	1.1	1.2
Test item	100 µg	13	1.0	13	3.1	1.1	1.1
Test item	316 µg	12	2.0	16	3.5	1.1	1.3
Test item	1000 µg	12	0.6	14	2.0	1.1	1.2
Test item	2500 µg	13	1.2	15	3.1	1.1	1.2
Test item	5000 µg	14	1.7	13	2.3	1.2	1.1
NaN3	10 µg	209	10.1	-	-	18.4	-
2-AA	2.5 µg	-	-	933	102.9	-	77.7
TA1537
Water	-	18	2.0	19	1.5	1.0	1.0
DMSO	-	19	2.1	20	2.1	1.0	1.0
Test item	31.6 µg	20	1.5	19	1.5	1.1	1.0
Test item	100 µg	19	2.1	19	1.7	1.0	0.9
Test item	316 µg	19	1.5	20	1.5	1.0	1.0
Test item	1000 µg	19	1.0	21	2.5	1.0	1.0
Test item	2500 µg	19	2.6	19	1.5	1.0	1.0
Test item	5000 µg	21	4.2	21	2.5	1.1	1.0
4-NOPD	40 µg	193	3.2	-	-	10.3	-
2-AA	2.5 µg	-	-	139	19.3	-	6.8
TA102
Water	-	314	44.0	258	46.0	1.0	1.0
DMSO	-	313	33.3	267	11.0	1.0	1.0
Test item	31.6 µg	319	27.5	257	18.5	1.0	1.0
Test item	100 µg	312	10.4	281	12.1	1.0	1.1
Test item	316 µg	315	7.4	275	31.5	1.0	1.0
Test item	1000 µg	324	24.6	215	34.5	1.0	0.8
Test item	2500 µg	311	7.8	231	21.4	1.0	0.9
Test item	5000 µg	324	16.0	185	10.0	1.0	0.7
MMS	1 µL	1148	94.2	-	-	3.7	-
2-AA	10 µg	-	-	1530	249.4	-	5.7

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

Treatment	Dose/plate	Revertant colonies per plate				Mutation factor
		Without S9		With S9
		Mean	SD	Mean	SD	-S9	+S9
TA98
Water	-	24	4.0	25	4.0	1.1	1.1
DMSO	-	22	5.9	23	5.3	1.0	1.0
Test item	31.6 µg	18	3.5	32	1.7	0.8	1.4
Test item	100 µg	26	8.6	27	3.5	1.1	1.2
Test item	316 µg	19	5.3	33	6.5	0.9	1.4
Test item	1000 µg	21	5.0	23	9.8	0.9	1.0
Test item	2500 µg	17	5.8	24	8.1	0.8	1.1
Test item	5000 µg	25	5.1	27	3.8	1.1	1.2
4-NOPD	10 µg	531	60.0	-	-	23.8	-
2-AA	2.5 µg	-	-	1125	7.0	-	48.9
TA100
Water	-	111	16.4	96	22.1	1.0	1.1
DMSO	-	107	16.9	90	21.6	1.0	1.0
Test item	31.6 µg	97	20.3	117	11.1	0.9	1.3
Test item	100 µg	126	21.6	103	9.6	1.2	1.2
Test item	316 µg	87	5.5	90	8.5	0.8	1.0
Test item	1000 µg	106	29.7	94	25.5	1.0	1.0
Test item	2500 µg	79	11.5	73	8.2	0.7	0.8
Test item	5000 µg	111	33.6	95	7.2	1.0	1.1
NaN3	10 µg	310	37.0	-	-	2.9	-
2-AA	2.5 µg	-	-	396	87.8	-	4.4
TA1535
Water	-	12	1.5	10	2.3	1.1	1.1
DMSO	-	11	1.5	10	2.5	1.0	1.0
Test item	31.6 µg	14	0.6	9	1.7	1.3	0.9
Test item	100 µg	12	2.1	12	3.5	1.1	1.2
Test item	316 µg	12	3.2	11	2.5	1.2	1.2
Test item	1000 µg	12	3.5	10	2.0	1.2	1.0
Test item	2500 µg	12	2.1	10	2.6	1.2	1.0
Test item	5000 µg	15	1.5	9	2.1	1.4	1.0
NaN3	10 µg	579	122.1	-	-	54.3	-
2-AA	2.5 µg	-	-	139	28.0	-	14.3
TA1537
Water	-	27	9.8	22	2.1	1.0	1.1
DMSO	-	26	7.2	20	3.2	1.0	1.0
Test item	31.6 µg	21	2.6	21	3.1	0.8	1.0
Test item	100 µg	24	0.6	21	2.6	0.9	1.0
Test item	316 µg	20	2.6	20	2.5	0.8	1.0
Test item	1000 µg	22	2.1	23	2.3	0.9	1.1
Test item	2500 µg	20	1.5	20	2.1	0.8	1.0
Test item	5000 µg	25	2.1	22	2.1	0.9	1.1
4-NOPD	40 µg	140	9.3	-	-	5.4	-
2-AA	2.5 µg	-	-	149	24.4	-	7.3
TA102
Water	-	268	22.0	295	20.7	1.0	0.9
DMSO	-	267	10.1	324	28.0	1.0	1.0
Test item	31.6 µg	276	9.1	306	20.8	1.0	0.9
Test item	100 µg	282	9.6	312	13.3	1.1	1.0
Test item	316 µg	251	6.6	285	12.8	0.9	0.9
Test item	1000 µg	284	9.1	287	15.0	1.1	0.9
Test item	2500 µg	248	15.7	278	21.1	0.9	0.9
Test item	5000 µg	263	24.3	278	25.4	1.0	0.9
MMS	1 µL	815	177.8	-	-	3.1	-
2-AA	10 µg	-	-	630	16.8	-	1.9

 

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

	TA98	TA100	TA1535	TA1537	TA102
Mean	25.5	94.1	16.7	10.5	283.3
SD	6.8	16.6	6.6	5.5	53.5
Min	11	49	4	3	142
Max	58	155	41	35	472
RSD (%)	26.5	17.6	39.4	51.9	18.9
n	1071	1258	1040	1035	819

 

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

	TA98	TA100	TA1535	TA1537	TA102
Substance Conc/plate	4-NOPD 10 µg	NaN3 10 µg	NaN3 10 µg	4-NOPD 40 µg	MMS 1 µL
Mean	440.0	650.2	875.3	107.3	1610.3
SD	162.5	219.1	289.3	37.3	546.3
Min	89	146	56	36	412
Max	1895	2493	1854	570	3437
RSD (%)	36.9	33.7	33.1	34.7	33.9
n	1077	1264	1049	1039	824

 

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

	TA98	TA100	TA1535	TA1537	TA102
Mean	29.3	95.5	13.3	10.7	345.7
SD	7.0	14.6	5.4	5.2	69.3
Min	15	60	3	3	157
Max	59	155	38	36	586
RSD (%)	23.8	15.3	40.4	48.1	20.0
n	1064	1252	1033	1028	812

 

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

	TA98	TA100	TA1535	TA1537	TA102
Substance Conc/plate	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 10 µg
Mean	1615.9	1533.5	167.7	221.6	841.2
SD	687.6	563.7	158.2	110.5	215.0
Min	70	169	23	23	310
Max	3287	3092	1954	888	3588
RSD (%)	42.5	36.8	94.3	49.8	25.6
n	1067	1254	1039	1031	815

Applicant's summary and conclusion

Conclusions:

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