Trisodium 5,5'-[(2-sulphonato-1,4-phenylene)bis(azo)]bis(salicylate)

Endpoint summary

Description of key information

The test item was evaluated for skin sensitisation using the DPRA (OECD 442C), KeratinoSens (OECD 442D) and h-CLAT (OECD 442E). A positive response was obtaind in the DPRA. However, negative findings were recorded in the KeratinoSens and the h-CLAT. Integrating all available results according to the "2-out-of-3" prediction model, the test item can be considered as a non-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:

2017-08-23 to 2017-09-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Version / remarks:

January 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

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 64.74%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

5.46

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	4835.2368	0.5340	5577.4585	0.5340
STD2	2314.6648	0.2670	2843.7996	0.2670
STD3	782.4597	0.1335	1440.4082	0.1335
STD4	525.4504	0.0667	706.2415	0.0667
STD5	266.4071	0.0334	357.4975	0.0334
STD6	135.9798	0.0167	180.2899	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine 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	1381.2087	0.1629	71.03	71.19	0.17	0.24
	1364.7275	0.1611	71.37
	1374.1948	0.1621	71.18
Test Item	0.0000	0.0109	100.00	100.00	0.00	0.00
	0.0000	0.0109	100.00
	0.0000	0.0109	100.00

Depletion of the Lysine Peptide

Lysine 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	2198.9507	0.2087	57.98	58.30	1.46	2.50
	2249.3828	0.2136	57.02
	2099.5659	0.1992	59.88
Test Item*	3684.4497	0.4641	7.12	5.46	1.61	29.50
	3755.0398	0.4731	5.34
	3811.9751	0.4802	3.91

* The peak observed in the lysine experiment could not be assigned clearly to lysine, as the retention time was delayed. Therefore, values for the delayed peak are given. However, they can only be considered as an estimation of the peptide depletion.

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

Categorization of the Test Item

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	--	--	--	100.00	High Reactivity	sensitizer
Positive Control	64.74	High Reactivity	sensitizer	71.19	Moderate Reactivity	sensitizer

Conclusions:

In this study under the given conditions the test item showed high reactivity towards the cysteine peptide. However, 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.

Executive summary:

In the present study the test item was dissolved in dist. water based on the results of the pre-experiments.

Based on a molecular weight of 552.36 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

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 the samples of the test item. Precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

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. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was noted for the samples of the positive control and turbidity was noted for the sample of the co-elution control of the positive control. 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 precipitation and phase separation was regarded as insignificant.

In the lysine peptide experiment no peak at the retention time of the lysine peptide (ca. 6.8 min) was observed. Although a peak was observed at a later retention time (ca. 7.7 min) which might represent the lysine peptide, unequivocal identification as the lysine peptide is not possible. Hence, evaluation of the lysine peptide depletion cannot be done and prediction model 2 should be considered. The sensitizing potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_{dist. water}).

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 64.74%. The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was >13.89% (100%). Based onprediction model 2 the test item might be considered as sensitiser. However, 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. The overall evaluation integrating the available in chemico and in vitro data for this endpoint is given in the enddpoint summary.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-21 to 2017-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by
addressing the second molecular key event of the adverse outcome pathway (AOP), namely
activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line
KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared
to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.17 in experiment 1; 2.99 in experiment 2).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

2.88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 1250 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

53.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

262.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 625 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

40.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC 1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of
64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	99.2	99.2	99.2	0.0
	8.00	104.1	110.9	107.5	4.8
	16.00	106.0	115.8	110.9	6.9
	32.00	111.4	117.1	114.3	4.0
	64.00	112.8	121.3	117.1	6.0
Test Item	0.61	99.2	105.3	102.2	4.3
	1.22	100.8	96.3	98.5	3.2
	2.44	99.0	89.5	94.3	6.7
	4.88	95.9	91.3	93.6	3.3
	9.77	92.8	79.0	85.9	9.7
	19.53	88.5	76.6	82.6	8.4
	39.06	82.8	66.2	74.5	11.7
	78.13	75.3	57.8	66.5	12.4
	156.25	66.2	50.4	58.3	11.2
	312.50	60.5	45.0	52.8	11.0
	625.00	57.3	40.4	48.9	11.9
	1250.00	53.8	45.3	49.5	6.0

Induction of Luciferase Activity – Overall Induction

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	1.34	1.04	1.19	0.21
	8.00	1.16	1.18	1.17	0.02
	16.00	1.66	1.46	1.56	0.15	*
	32.00	2.16	1.98	2.07	0.13	*
	64.00	3.17	2.99	3.08	0.13	*
Test Item	0.61	1.67	1.07	1.37	0.42
	1.22	1.52	1.05	1.28	0.33
	2.44	1.36	1.17	1.26	0.14
	4.88	1.19	1.00	1.09	0.14
	9.77	1.29	0.91	1.10	0.27
	19.53	1.03	0.91	0.97	0.09
	39.06	1.01	0.82	0.92	0.13
	78.13	1.02	0.83	0.93	0.14
	156.25	1.12	0.82	0.97	0.22
	312.50	1.68	1.01	1.34	0.47
	625.00	2.86	1.30	2.08	1.10
	1250.00	2.88	1.26	2.07	1.14

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

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	262.80	n/a	262.80	-
Imax	2.88	1.30	2.09	1.12
IC30[µM]	123.60	31.92	77.76	64.83
IC50[µM]	n/a	167.17	-	-

n/a not applicable

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	15.8	pass	11.6	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass
EC1.5 PC	7 < x < 34 µM	13.42	pass	17.36	pass
Induction PC at 64 µM	2.00 < x < 8.00	3.17	pass	2.99	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might be considered as non-sensitiser.
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.

Executive summary:

In the present study the test item was dissolved in dist. water. The maximum solubility was determined as 125 mM. Based on a molecular weight of 552.36 g/mol a stock solution of 125 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

1250, 625, 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88, 2.44, 1.22, 0.61 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction in a concentration range with viabilities >70%. Under the condition of this study the test item might be considered as non-sensitiser.

The overall evaluation integrating the available in chemico and in vitro data for this endpoint is given in the enddpoint summary.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-09 to 2017-11-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Details on the study design:

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 sensitisers and
non-sensitisers.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (449% experiment 1; 406% experiment 2) and 200% for CD54 (443% experiment 1; 272% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

94

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 167.45 µM

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

145

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 347.22 µM

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentrations: 200.94 and 347.22 µM

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 500 µM

Other effects / acceptance of results:

Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

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	-	96.2	96.3	96.6	1040	934	624	416	310	100	100	167	150
Solvent Control	0.20%	96.2	95.7	96.1	1124	1000	626	498	374	120	121	180	160
DNCB	4.00	81.9	81.5	80.6	2946	2368	712	2234	1656	449	443	414	333
Aluminium Orange RL pc-w	500.00	93.4	94.0	94.0	932	1059	637	295	422	71	136	146	166
	416.67	94.2	93.3	93.7	924	1075	673	251	402	60	130	137	160
	347.22	94.6	94.2	94.4	952	1103	653	299	450	72	145	146	169
	289.35	95.0	94.3	94.2	969	1109	686	283	423	68	136	141	162
	241.13	94.6	94.9	94.7	942	1037	651	291	386	70	125	145	159
	200.94	94.7	93.5	94.4	1005	1049	637	368	412	88	133	158	165
	167.45	94.9	95.6	95.6	1034	1060	644	390	416	94	134	161	165
	139.54	95.2	95.3	95.0	991	1053	643	348	410	84	132	154	164

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	-	97.3	96.5	96.0	1509	1180	652	857	528	100	100	231	181
Solvent Control	0.20%	96.3	94.3	96.2	1588	1231	673	915	558	107	106	236	183
DNCB	4.0	87.4	85.8	85.5	4346	2147	632	3714	1515	406	272	688	340
Aluminium Orange RL pc-w	500.00	93.7	94.1	93.0	1296	1216	709	587	507	68	96	183	172
	416.67	93.0	92.4	91.3	1366	1194	705	661	489	77	93	194	169
	347.22	92.7	91.7	90.7	1397	1191	725	672	466	78	88	193	164
	289.35	91.8	91.9	92.2	1343	1178	716	627	462	73	88	188	165
	241.13	94.5	94.6	94.2	1120	1163	707	413	456	48	86	158	164
	200.94	94.4	93.3	94.1	1400	1205	728	672	477	78	90	192	166
	167.45	93.6	94.1	93.9	1368	1200	717	651	483	76	91	191	167
	139.54	94.8	94.4	94.8	1318	1138	688	630	450	74	85	192	165

 

Acceptance Criteria

Acceptance Criterion	range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability medium and solvent control [%]	>90	95.7	-	96.6	pass	94.3	-	97.3	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	449			pass	406			pass
RFI of positive control of CD54	≥200	443			pass	272			pass
RFI of solvent control of CD86	<150	120			pass	107			pass
RFI of solvent control of CD54	<200	121			pass	106			pass
MFI ratio IgG1/CD86 for medium control [%]	>105	167			pass	231			pass
MFI ratio IgG1/CD86 for solvent control [%]	>105	180			pass	236			pass
MFI ratio IgG1/CD54 for medium control [%]	>105	150			pass	181			pass
MFI ratio IgG1/CD54 for solvent control [%]	>105	160			pass	183			pass

Historical Data

Criterion	mean	SD	N
cell viability solvent controls [%]	97.0	1.3	672
number of test doses with viability >50%	-	-	1786
RFI of positive control of CD86	401.0	146.8	112
RFI of positive control of CD54	576.6	312.0	112
RFI of solvent control of CD86	115.0	15.1	112
RFI of solvent control of CD54	118.8	25.5	112
MFI ratio IgG1/CD86 for medium control [%]	202.4	50.0	112
MFI ratio IgG1/CD86 for DMSO control [%]	221.6	58.5	112
MFI ratio IgG1/CD54 for medium control [%]	141.0	24.7	112

 

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.
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.

Executive summary:

In the present study the test item was dissolved in 0.9% NaCl. The maximum solubility was determined as 50 mg/mL. For the dose finding assay stock solutions with concentrations ranging from 50.00 mg/mL to 0.39 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. Since the maximum solubility was reached, the main experiment was performed covering the following concentration steps:

500.00, 416.67, 347.22, 289.35, 241.13, 200.94, 167.45 and 139.54 µg/mL

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 reduced to 93.4% (CD86), 94.0% (CD54) and 94.0% (isotype IgG1 control) in the first experiment and to 93.7% (CD86), 94.1% (CD54) and 93.0% (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 might be considered as non-sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (449% experiment 1; 406% experiment 2) and 200% for CD54 (443% experiment 1; 272% experiment 2) were clearly exceeded.

The overall evaluation integrating the available in chemico and in vitro data for this endpoint is given in the enddpoint summary.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

For skin sensitisation, the "2-out-of-3" prediction model (PM) according to Bauch et al. (2012), is the most commonly used and accepted PM to integrate the outcome of individual DPRA, KeratinoSens and h-CLAT assays for a prediction of the in vivo effect of respective test substances. Using this PM, a substance is assigned as skin sensitiser if the results obtained in at least 2 of the above 3 assays are positive. In case of the discussed test item the DPRA showed protein bindig potential. However, both the KeratinoSens and the h-CLAT did not reveal indications for a skin sensitisation potential. Applying the "2-out-of-3" PM the discussed test item can therefore be regarded as non-sensitiser to skin also under in vivo conditions. Based here upon, the discussed test item is not subject for labelling and classification requirements regarding this endpoint.