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

Endpoint summary

Description of key information

OECD Guideline in vitro/ in chemico studies have been performed to address each of the key events of skin sensitisation (molecular interaction with skin proteins, inflammatory response in keratinocytes and activation of dendritic cells). The results of these tests are considered valid and all tests concluded that balsalazide acid is not a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th June 2018 - 19th July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”.
Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP

Specific details on test material used for the study:

Batch No.: 0400
Purity: 96%
Appearance: Orange solid
Storage conditions: Room temperature

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.83%.

Key result

Run / experiment:

other: Test item

Parameter:

other: Mean peptide depletion (%)

Value:

2.41

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Reference controls

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes: The mean depletion of both peptides was 64.83%.

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. The test item was soluble in dist. water : acetonitrile 1:1 (v/v). No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using dist. water : acetonitrile 1:1 (v/v). All test item solutions were freshly prepared immediately prior to use

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant

Co-elution with the Peptide Peaks

No relevant co-elution of the test item with any of the peptide peaks was observed

Depletion of the cysteine peptide depletion:

Cysteine peptide
Sample	Peak area at 220 nm	Peptide Conc. [mM]	Peptide depletion [%]	Mean peptide depletion [%]	SD of peptide depletion [%]	CV of peptide depletion [%]
Positive control	4.7290 4.7400 4.7210	0.1451 0.1454 0.1449	71.19 71.12 71.24	71.18	0.06	0.08
Test item	16.1620 16.0220 15.7820	0.4951 0.4908 0.4835	2.31 3.15 4.60	3.35	1.16	34.65

Depletion of the Lysine peptide

Cysteine peptide
Sample	Peak area at 220 nm	Peptide Conc. [mM]	Peptide depletion [%]	Mean peptide depletion [%]	SD of peptide depletion [%]	CV of peptide depletion [%]
Positive control	6.2940 5.9870 5.6530	0.2177 0.2071 0.1955	56.28 58.41 60.73	58.47	2.23	3.81
Test item	14.2650 14.3900 14.3550	0.4938 0.4981 0.4969	1.95 1.10 1.34	1.46	0.44	30.32

Categorization of the test item:

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

Prediction model	Prediction model 1 (Cysteine peptide and lysine peptide/ratio 1:10 and 1:50)			Prediction model 2 (cysteine peptide/test item ratio: 1: 10)
Test substance	Mean peptide depletion [%]	Reactivity category	Prediction	Mean peptide depletion [%]	Reactivity category	Prediction
Test item	2.41	Minimal reactivity	No sensitiser	3.35	Minimal reactivity	No sensitiser
Positive control	64.83	High reactivity	Sensitiser	71.18	Moderate reactivity	sensitiser

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given conditions of the study, the test item showed minimal reactivity towards both peptides. Therefore, the test item, balsalazide acid, might be considered as a non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2018 - 04 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 09 October 2017

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Batch no: 0400

Details on the study design:

Skin sensitisation (In vitro test system)

Key result

Run / experiment:

other: CD86 upregulation (%)

Parameter:

other: dendritic cell activation

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD54 upregulation (%)

Parameter:

other: dendritic cell activation

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No cytotoxic effects were observed for the cells treated with the test item.

ACCEPTANCE OF RESULTS: See Table 6

Reactivity check of the cell stock:

Doubling time of the cells was monitored and found to be 39.9 h and 39.4 h which are within the doubling time range specified by the manufacturer (35 - 50 h).

 

Table 1:  Results of the Cell Batch 1 Activation Test

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	87.5	373	>150	88.1	358	>200	yes	pass
NiSO4	100 µg/mL	83.5	295	>150	82.1	603	>200	yes	pass
LA	1000 µg/mL	95.7	81	£150	95.8	100	£200	no	pass

 

Table 2:  Results of the Cell Batch 21 Activation Test

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	85.5	251	>150	84.7	287	>200	yes	pass
NiSO4	100 µg/mL	74.9	249	>150	75.5	518	>200	yes	pass
LA	1000 µg/mL	94.8	67	£150	95.0	108	£200	no	pass

 

The positive controls DNCB and NiSO₄led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.

The cell batch was accepted for further testing.

 

Solvent Finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 250 mg/mL.

 

Dose Finding Assay

The dose finding assay was performed using stock solutions with a concentration of 500 mg/mL (applied concentration 1000 µg/mL).

Table 3:  Results of the Dose Finding Assay

Sample		Experiment 1
		Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	95.90
Solvent Control	DMSO	--	95.30
Balsalazide acid	C8	7.81	95.20
	C7	15.63	94.60
	C6	31.25	93.90
	C5	62.50	95.00
	C4	125.00	94.80
	C3	250.00	95.30
	C2	500.00	95.50
	C1	1000.00	95.30
Calculated CV75 [µg/mL]		No CV75
Mean CV75 [µg/mL]		No CV75
SD CV 75 [µg/mL]		No SD

 

The dose finding assay was performed in one independent run. Since there was no cytotoxicity, no CV75 could be derived. The main experiment was performed covering a concentration range from 1000 – 279.08 µg/mL (500 – 139.54 mg/mL stock solution).

 

Results CD54 and CD86 Expression

For determination of the cell surface markers CD54 and CD86 two independent experiments were performed using separate cultivated cells at passage 10 (first experiment) and 12 (second experiment). For each experiment separately weighted samples and preparations were used.

Table4:  CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	94.3	95.8	95.8	1679	1010	543	1136	467	95	96	309	186
Solvent Control	0.20%	95.3	95.6	95.5	1737	1030	546	1191	484	100	100	318	189
DNCB	4.00	82.5	82.4	81.4	4206	2308	601	3605	1707	303	353	700	384
Balsalazide acid	500	95.7	95.9	95.4	1611	1219	598	1013	621	85	128	269	204
	416.67	95.6	95.9	95.8	1658	1230	602	1056	628	89	130	275	204
	347.22	95.1	95.2	95.4	1787	1219	592	1195	627	100	130	302	206
	289.35	94.8	95.3	95.7	1793	1211	595	1198	616	101	127	301	204
	241.13	95.0	95.6	95.8	1875	1231	587	1288	644	108	133	319	210
	200.94	95.8	96.0	96.3	1770	1114	558	1212	556	102	115	317	200
	167.45	95.1	95.1	95.2	1803	1046	548	1255	498	105	103	329	191
	139.54	95.7	95.6	95.6	1575	1082	547	1028	535	86	111	288	198

 

Table5: CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	96.1	96.1	95.5	1375	904	547	828	357	97	89	251	165
Solvent Control	0.20%	96.6	96.7	97.0	1396	945	544	852	401	100	100	257	174
DNCB	4.0	84.0	85.0	85.1	4295	2175	553	3742	1622	439	404	777	393
Balsalazide acid	500.00	97.0	96.6	97.1	1483	1161	612	871	549	102	137	242	190
	416.67	96.6	97.1	96.9	1601	1120	606	995	514	117	128	264	185
	347.22	96.7	97.4	97.3	1535	1100	596	939	504	110	126	258	185
	289.35	97.2	96.9	96.9	1719	1093	570	1149	523	135	130	302	192
	241.13	96.6	96.2	96.8	1523	1123	590	933	533	110	133	258	190
	200.94	96.7	96.9	96.4	1790	1067	571	1219	496	143	124	313	187
	167.45	96.4	96.9	96.9	1574	1055	569	1005	486	118	121	277	185
	139.54	96.9	96.8	96.5	1835	1096	592	1243	504	146	126	310	185

 

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study Balsalazide acid was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500.00 mg/mL to 139.54 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

In all experiments no precipitation or turbidity of the test item was observed for all the concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was 95.7% (CD86), 95.9% (CD54) and 95.4% (isotype IgG1 control) in the first experiment and 97.0% (CD86), 96.6% (CD54) and 97.1% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item can be considered as non-sensitiser.

The controls confirmed the validity of the study for all experiments as shown in Table 6.

 

Table 6:  Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	94.3	-	95.8	pass	95.5	-	97.0	pass
number of test dosed with viability >50% CD86	≥4	8			pass	8			pass
number of test dosed with viability >50% CD54	≥4	8			pass	8			pass
number of test dosed with viability >50% IgG1	≥4	8			pass	8			pass
RFI of positive control of CD86	≥150	303			pass	439			pass
RFI of positive control of CD54	≥200	353			pass	404			pass
RFI of solvent control of CD86	<150	105			pass	103			pass
RFI of solvent control of CD54	<200	104			pass	112			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	309			pass	251			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	318			pass	257			pass
MFI ratio CD54/IgG1for medium control [%]	>105	186			pass	165			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	189			pass	174			pass

 

 

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore, the test item might be considered as non-sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th June 2018 - 30th July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line (e.g. KeratinoSens™) derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS category 1.

Specific details on test material used for the study:

Batch No.: 0400
Purity: 96%
Appearance: Orange solid
Storage conditions: Room temperature

Key result

Run / experiment:

other: Experiments 1 and 2

Parameter:

other: Luciferase induction

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Solvent control

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No dose response for luciferase activity induction was observed.

Other effects / acceptance of results:

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes: Number of positive control concentration steps with significant luciferase activity induction >1.5: 2.0 (Expt 1) and 3.0 (Expt 2); EC1.5: 18.06 (Expt 1) and 9.77 (Expt 2); Induction at 64 µM: 5.22 (Expt 1) and 5.60 (Expt 2)
- Acceptance criteria met for solvent control: Yes: CV: 15.1% (Expt 1) and 8.9 (Expt 2)

Table 1: Induction of Luciferase activity experiment 1:

Experiment 1	Concentration [μM]	Fold induction					Significance
		Rep 1	Rep 2	Rep 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00	1.25	1.27	1.23	1.25	0.02
	8.00	1.28	1.25	1.58	1.37	0.18
	16.00	1.33	1.40	1.47	1.40	0.07
	32.00	2.02	2.03	2.47	2.17	0.25	*
	64.00	4.42	5.04	6.19	5.22	0.90	*
Test item	0.98	0.98	0.95	1.03	0.99	0.04
	1.95	1.07	0.86	0.80	0.91	0.14
	3.91	0.92	0.82	0.78	0.84	0.07
	7.81	1.09	0.74	0.88	0.91	0.18
	15.63	1.11	0.88	0.78	.92	0.17
	31.25	0.85	0.79	0.78	0.81	0.03
	62.50	0.77	0.65	0.71	0.71	0.06
	125.00	0.76	0.66	0.70	0.70	0.05
	250.00	0.90	0.59	0.65	0.72	0.17
	500.00	0.92	0.56	0.62	0.70	0.19
	1000.00	0.63	0.53	0.56	0.57	0.05
	2000.00	0.47	0.48	0.51	0.49	0.02

* = significant induction according to Student’s t-test, p<0.05

Table 2: Induction of Luciferase activity experiment 2:

Experiment 2	Concentration [μM]	Fold induction					Significance
		Rep 1	Rep 2	Rep 3	Mean	SD
Solvent control	-	1.00	1.00	1.00	1.00	0.00
Positive control	4.00	1.26	1.26	1.31	1.28	0.03
	8.00	1.43	1.34	1.45	1.40	0.06
	16.00	1.92	1.63	1.95	1.83	0.18	*
	32.00	2.37	2.06	2.52	2.32	0.24	*
	64.00	6.03	4.59	6.19	5.60	0.88	*
Test item	0.98	2.35	0.95	0.98	1.43	0.80
	1.95	0.98	0.99	1.05	1.01	0.04
	3.91	1.03	1.08	1.04	1.05	0.03
	7.81	1.12	0.96	1.16	1.08	0.10
	15.63	1.06	1.14	1.24	1.15	0.09
	31.25	1.12	1.04	1.13	1.10	0.05
	62.50	1.07	1.06	1.11	1.08	0.02
	125.00	1.02	0.99	0.94	0.98	0.04
	250.00	0.91	0.84	0.92	0.89	0.04
	500.00	0.88	0.66	0.88	0.81	0.13
	1000.00	0.72	0.65	0.67	0.68	0.03
	2000.00	0.52	0.53	0.65	0.57	0.07

Table 3: Induction of luciferase activity - overall induction

	Concentration [μM]	Fold induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent control	-	1.00	1.00	1.00	0.00
Positive control	4.00	1.25	1.28	1.26	0.02
	8.00	1.37	1.40	1.39	0.02
	16.00	1.40	1.83	1.62	0.31
	32.00	2.17	2.32	2.24	0.10	*
	64.00	5.22	5.60	5.41	0.27	*
Test item	0.98	0.99	1.43	1.21	0.31
	1.95	0.91	1.01	0.96	0.07
	3.91	0.84	1.05	0.95	0.15
	7.81	0.91	1.08	0.99	0.12
	15.63	0.92	1.15	1.03	0.16
	31.25	0.81	1.10	0.95	0.20
	62.50	0.71	1.08	0.89	0.26
	125.00	0.70	0.98	0.84	0.20
	250.00	0.72	0.89	0.80	0.12
	500.00	0.70	0.81	0.76	0.08
	1000.00	0.57	0.68	0.63	0.07
	2000.00	0.49	0.57	0.53	0.06

* = significant induction according to Student’s t-test, p<0.05

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given conditions the test item, balsalazide acid did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as a non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Balsalazide acid is not a skin sensitiser based on the negative results of the three recommended in vtro/ in chemico OECD Guideline skin sensitisation studies.