reaction mass of diethyl (E)-2-methylbut-2-enedioate and diethyl (Z)-2-methylbut-2-enedioate

Endpoint summary

Description of key information

In in silico and in vitro tests the test substance was determined to be peptide reactive (OECD 442C) and to activate keratinocytes (OECD 442D), wheras the test substance did not activate dendritic cells (OECD 442E). The weight of evidence conclusion from the in vitro test battery is that the test substance is considered to be a skin sensitiser.

The test substance (1.1%) indicated no potential for dermal irritation or allergic contact sensitisation in a human repeated insult patch test. This leads to a NESIL of 606 µg/cm² and therefore the sub-category is 1B.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 September 2016 - 06 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

2016

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

Principles of method if other than guideline:

In addition, the h-CLAT was performed according to the methods described in the following publications:
- Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol In Vitro. 2012 Oct;26(7):1150-60.
- Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. (2010). A comparative evaluation of in vitro skin sensitization tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010 Aug;38(4):275-84.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Skin sensitisation (In vitro test system)
Cell line used: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202.

Technical material and conditions:
- Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 µg/mL of penicillin and 100 μg/mL of streptomycin).
- FACS buffer: PBS with 0.1% (w/v) BSA
- Antibodies: Anti - CD86 antibody (Clone: Fun-1), Anti – CD54 antibody (Clone: 6.5B5),
FITC labelled-mouse IgG1 (isotype control)

Controls used:
- Vehicle control: DMSO (final concentration 0.2%)
- Positive control: DNCB in DMSO (diluted with culture medium to a final concentration of 2 and 3 μg/mL DNCB)
- Isotype control: mouse IgG1.

Test procedure:

Dose Finding Assay, XTT Test:
Two independent cytotoxicity experiments were performed on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). All dose groups (eight concentrations) were tested in 7 replicates for each XTT test. The incubation period was 24 ± 0.5 hours. After incubation with the XTT labelling mixture the absorbance was measured at 450 nm.

Human Cell Line Activation Test (h-CLAT):
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry.

Acceptance criteria:
- Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- In the DMSO solvent control, RFI values compared to the medium control of both
CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- For the test item resulting in negative outcome, the cell viability at the 1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test item are accepted independent by the cell viability.)
- The cell viability of at least 4 doses in each experiment should be ≥50%.

Evaluation of results:
The test item is tested in 2 independent runs. The test item is considered to be a sensitizer
- if the RFI of CD86 is ≥ 150% in both independent run data or
- if the RFI of CD54 is ≥ 200% in both independent run data.
Otherwise it is considered to be a non-sensitiser.
In case of different results in both runs, a third run has to be performed. The test item is considered to be a sensitizer:
- if the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data or
- if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data.
Otherwise it is considered to be a non-sensitiser.

Positive control results:

The positive controls exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Key result

Run / experiment:

other: First run - concentrations: 274, 328, 394, 473, 567 and 681 µg/mL

Parameter:

other: RFI (%) CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: First run - concentration: 817 µg/mL

Parameter:

other: RFI (%) CD54

Value:

167.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: excluded from the evaluation, due to observed precipitations

Run / experiment:

other: First run - concentration: 980 µg/mL

Parameter:

other: RFI (%) CD54

Value:

295.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: excluded from the evaluation, due to observed precipitations

Key result

Run / experiment:

other: Second run - concentrations: 274, 328, 394, 473, 567 and 681 µg/mL

Parameter:

other: RFI (%) CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: Second run - concentration: 817 µg/mL

Parameter:

other: RFI (%) CD86

Value:

258.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: excluded from the evaluation, due to observed precipitations

Run / experiment:

other: Second run - concentration: 980 µg/mL

Parameter:

other: RFI (%) CD86

Value:

1 192.8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: excluded from the evaluation, due to observed precipitations

Table 1: Results of the first h-CLAT run for the Test Item

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	470.9*	63.9
		CD 86	371.7*
	3.0	CD 54	570.9*	67.9
		CD 86	457.2*
Test Item	274	CD 54	54.9	90.5
		CD 86	56.6
	328	CD 54	87.8	96.4
		CD 86	60.5
	394	CD 54	93.9	93.5
		CD 86	88.8
	473	CD 54	95.1	86.0
		CD 86	100.0
	567	CD 54	97.6	85.5
		CD 86	94.7
	681	CD 54	129.3	83.3
		CD 86	125.0
	817^P	CD 54	167.1	61.9
		CD 86	258.6*
	980^P	CD 54	295.1*	22.6
		CD 86	1192.8*

P: Precipitation, *RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Table 2: Results of the second h-CLAT run for the Test Item

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	261.6*	70.7
		CD 86	425.1*
	3.0	CD 54	372.8*	67.0
		CD 86	383.6*
Test Item	274	CD 54	13,4.4	95.5
		CD 86	110.1
	328	CD 54	143.0	99.3
		CD 86	116.2
	394	CD 54	158.1	97.1
		CD 86	136.5
	473	CD 54	153.8	96.1
		CD 86	132.4
	567	CD 54	166.7	87.6
		CD 86	110.8
	681	CD 54	176.3	86.5
		CD 86	125.7
	817^P	CD 54	168.8	79.4
		CD 86	168.2*
	980^P	CD 54	251.6*	40.5
		CD 86	550.7*

P: Precipitation, *RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered not to be a skin sensitiser up to a test item concentration of 681 μg/mL, under the test conditions of this study.

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h- CLAT). The test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 274, 328, 394, 473, 567, 681, 817 and 980μg/mL. The test substance was administered to THP-1 cells for 24 ± 0.5 hours. The RFI of CD86 was greater than 150% in the two highest tested test item concentrations and the RFI of CD54 was greater than 200% in the highest tested test item concentrations in both h-CLAT runs. The two highest tested concentrations (817 and 980μg/mL) were excluded from the evaluation in both runs, as precipitations were observed. Considering this, the test item is considered to be a non-sensitiser under the test conditions of this study. The controls confirmed the validity of the study.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 February 2017 - 04 July 2017

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)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system)
Cell line used: KeratinoSensTM (Givaudan, Switzerland)

Technical material and conditions:
- Maintenance Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS + 1 % Geneticin (final concentration: 500 µg/mL)
- Assay Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS
- Test Item Exposure Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 1 % FBS
- Luciferace reagent: Luciferase Assay Substrate (Promega, Cat. No.: E1501)
- Assay Buffer: Luciferase Assay Buffer (Promega, Cat. No.: E1501)
- Lysisbuffer: Luciferase Cell Culture Lysis (Promega Cat. No.: E1531)
- MTT Solution: MTT stock solution: 5 mg/mL MTT in DPBS
- SDS solution: 10% (wlv) SDS in dist. water
- DPBS solution: DPBS solution (without Ca2+/Mg2+)

Controls used:
- Vehicle control: DMSO: 1% (vlv) in test item exposure medium
- Positive control: Cinnamic aldehyde in DMSO, 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
- Blank control: vehicle control without cells

Test procedure:
Each concentration step of the test item (twelve concentrations) and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates.
The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a plate reader.
Cell viability was determined by a MTT assay. The test substance was incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 10% SDS solution the plate was incubated at 37 °C ± 1 °C and 5% CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at A = 600 nm.

Acceptance criteria:
- 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.

Evaluation of results:
The test item is considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.

Positive control results:

The results of the positive control were within the historical control range.

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 (µM)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5 (µM)

Value:

277.16

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: EC1.5 (µM)

Value:

283.63

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: Experiment 1 - 30.13 µM (lowest tested concentration with a significant Imax)

Parameter:

other: Imax

Value:

1.52

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2 - 482 µM (lowest tested concentration with a significant Imax)

Parameter:

other: Imax

Value:

1.76

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 3 - 482 µM (lowest tested concentration with a significant Imax > 1.5)

Parameter:

other: Imax

Value:

1.64

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not applicable
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

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	1.20	1.20	1.22	1.21	0.01
	8.00	1.20	1.23	1.29	1.24	0.05
	16.00	1.30	1.25	1.56	1.37	0.17
	32.00	1.86	1.58	2.28	1.90	0.35	*
	64.00	2.86	2.81	2.81	2.83	0.03	*
Test Item	0.94	[1.35]	[1.85]	[1.65]	[1.61]^a)	[0.25]
	1.88	1.10	1.39	1.40	1.30	0.17
	3.77	1.17	1.51	1.40	1.36	0.18
	7.53	1.47	1.35	1.65	1.49	0.15
	15.06	1.43	1.16	1.36	1.32	0.14
	30.13	1.41	1.27	1.89	1.52	0.33	*
	60.25	1.69	1.38	1.61	1.56	0.16	*
	120.50	1.58	1.66	1.79	1.67	0.10	*
	241.00	2.05	1.79	2.10	1.98	0.16	*
	482.00	2.16	2.01	2.09	2.09	0.07	*
	964.00	2.77	2.28	2.53	2.53	0.25	*
	1928.00	2.58	2.63	2.98	2.73	0.22	*

* significant induction according to Student’s t-test, p<0.05,^a) The lowest concentration was excluded from further evaluation, since an increased fold induction >1.5 was observed, which was not dose dependent and, therefore, the value was regarded as outlier.

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	1.08	1.04	0.99	1.04	0.04
	8.00	1.20	1.17	1.15	1.17	0.02
	16.00	1.20	1.24	1.25	1.23	0.03
	32.00	1.85	1.55	1.86	1.75	0.18	*
	64.00	3.53	3.07	3.82	3.48	0.38	*
Test Item	0.94	1.10	0.97	0.83	0.97	0.13
	1.88	1.13	0.95	0.89	0.99	0.13
	3.77	1.19	1.02	0.90	1.04	0.15
	7.53	1.09	1.11	0.87	1.03	0.13
	15.06	1.17	1.04	0.93	1.05	0.12
	30.13	1.23	1.11	1.02	1.12	0.11
	60.25	1.32	1.16	1.06	1.18	0.13
	120.50	1.45	1.30	1.17	1.31	0.14
	241.00	1.66	1.37	1.33	1.45	0.18
	482.00	1.79	1.94	1.56	1.76	0.19	*
	964.00	1.84	2.11	2.04	2.00	0.14	*
	1928.00	2.27	2.13	2.37	2.26	0.12	*

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

Table 3: Induction of Luciferase Activity Experiment 3

Experiment 3	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	1.04	1.09	1.05	1.06	0.03
	8.00	1.13	1.24	0.97	1.11	0.13
	16.00	1.18	1.34	1.18	1.23	0.09
	32.00	1.67	2.26	2.12	2.02	0.31	*
	64.00	3.68	4.04	3.98	3.90	0.19	*
Test Item	0.94	0.89	1.57	1.00	1.15	0.37
	1.88	0.89	1.37	0.97	1.08	0.26
	3.77	0.87	1.86	0.81	1.18	0.59
	7.53	0.86	1.26	0.74	0.95	0.28
	15.06	0.97	1.62	1.01	1.20	0.37
	30.13	0.92	1.43	0.88	1.07	0.31
	60.25	0.95	1.55	0.90	1.14	0.36
	120.50	1.13	1.78	1.19	1.36	0.36
	241.00	1.39	1.86	1.15	1.47	0.36
	482.00	1.36	1.96	1.61	1.64	0.30	*
	964.00	1.82	2.65	1.41	1.96	0.63	*
	1928.00	1.93	2.65	1.88	2.16	0.43	*

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

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.

Executive summary:

The keratinocyte activating potential of test substance was evaluated in the LuSens Assay (1928, 964.0, 482.0, 241.0, 120.5, 60.25, 30.13, 15.06, 7.53, 3.77, 1.88, and 0.94 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.

In the first experiment, a max luciferase activity(I_max)induction of 2.73 was determined at a test item concentration of 1928 µM. The corresponding cell viability was 146.8%. The lowest tested concentration with a significant luciferase induction >1.5 (1.52) was found to be 30.13 µM. The corresponding cell viability was >70% (97.4%). No EC_1.5could be calculated.

In the second experiment, a max luciferase activity (l_max) induction of 2.26 was determined at a test item concentration of 1928 µM. The corresponding cell viability was 100.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.76) was found to be 482 µM. The corresponding cell viability was >70% (92.5%). The calculated EC_1.5was < 1000 µM (277.16 µM).

In the third experiment, a max luciferase activity (l_max) induction of 2.16 was determined at a test item concentration of 1928 µM. The corresponding cell viability was 94.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.64) was found to be 482 µM. The corresponding cell viability was >70% (99.4%). The calculated EC_1.5was < 1000 µM (283.63 µM).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

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

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 September 2016 - 23 May 2017

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))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system)

Synthetic peptides used:
- Synthetic peptides containing cysteine: Ac-RFAACAA-OH, AnaSpec, Batch number: 1556171
- Synthetic peptides containing lysine: Ac-RFAAKAA-OH, AnaSpec, Batch number: 1556172

Controls used:
- Stability controls: 0.5 mM cysteine or lysine peptide in acetonitrile
- Precision Controls: 0.5 mM cysteine or lysine peptide in acetonitrile
- Positive control: 100 mM Cinnamic aldehyde in acetonitrile
- Co-elution control: 5 mM test item or positive control in buffer solution

Test substance:
- 100 mM test substance in acetonitrile.

Peptide stock solution:
- 0.667 mM cystein or lysine in the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Depletion Samples:
- Cysteine Peptide Depletion Samples: Acetonitrile solutions of test substance or positive control diluted with the Cysteine
Peptide, final concentration: 5 mM test substance or positive control and 0.5 mM Cysteine
- Lysine Peptide Depletion Samples: Acetonitrile solutions of test substance or positive control diluted with the Lysine Peptide, final concentration: 25 mM test substance or positive control 0.5 mM Lysine

Incubation:
- ≥ 22 h, 25 °C

HPLC conditions:
- HPLC: Waters Alliance 2695 separation module and2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5m, 100  2.1 mm
- Guard column: Phenomenex AJO4286
- Column temperature: 30 C
- Sample temperature: 25 C
- Mobile Phase (MP) A: 0.1% TFA in Water
- Mobile Phase (MP) B: 0.085% TFA in ACN
Gradient: Time (minutes): 0 min: 90% MP A, 20 min: 75 % MP A, 21 min: 10 % MP A, 23 min: 10 % MP A, 23.5 min: 90% MP A, 30 mi: 90 % MP A
- Flow rate: 0.35 mL/minute
- Stroke volume 25 μL
- Detector wavelength: UV, 220 nm
- Injection volume: 2 μL (slow draw rate)
- Run time: 30 minutes
- Approximate retention time (Cysteine) 11 minutes
- Approximate retention time (Lysine) 7 minutes

Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at A = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions.

Acceptance criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r2 > 0.99,
- the mean positive control is between 60.8% and 100% for the cysteine peptide and the coefficient of variation (CV) is < 14.9%,
- the positive control depletion is between 40.2% and 69.0% for the lysine peptide and the coefficient of variation (CV) is < 11.6%,
- the mean peptide concentration of the reference controls is between 0.45 and 0.55 mM and the coefficient of variation (CV) < 14.9% (cysteine) and < 11.6% (lysine).
- the coefficient of variation (CV) of peptide peak areas for the test item is < 14.9% (cysteine) and < 11.6% (lysine).

Evaluation of results:
The Cysteine depletion model was used for evaluation of results. The test item was considered positive to be a skin sensitiser, if the cystein peptide depletion was > 13.89 % and negative to be a skin sensitiser, if the cystein peptide depletion was ≤ 13.89

Positive control results:

The acceptance criteria for the positive control were met.

Key result

Run / experiment:

mean

Parameter:

other: Cystein depletion (%)

Value:

29.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

mean

Parameter:

other: Lysine depletion (%)

Value:

14.7

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: The acceptance criteria for the lysine peptide assay was not met as column overload of the test item caused chromatographic interference resulting in non-resolution of the Lysine peak so invalidating the results from the assay.

Other effects / acceptance of results:

OTHER EFFECTS:
- Column overload of the test items caused chromatographic interference resulting in non-resolution of the Lysine peak.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Applying the following Cysteine depletion model, the reactivity of the test item is classed as moderate and the DPRA prediction is positive. Therefore the test item is considered to be a potential skin sensitizer.

Executive summary:

The solubility of test item in acetonitrile at a nominal concentration of 100 mM was confirmed. Analysis of solutions of test item in the Lysine containing peptide was inconclusive due to column overload causing the Lysine peak to be unresolved and co-elute. Apart from the test item coefficient of variation in the Lysine peptide all analytical acceptance criteria were met. Therefore the DPRA prediction and the reactivity of test item was based on the mean and the individual depletion values in the cysteine peptide only. The result from the Cysteine assay places the test item in the reactivity class of “moderate reactivity” hence the test item is predicted as a potential skin sensitizer by DPRA.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a test battery of non in vivo experiments was performed, as a single in vitro/in chemico test is not sufficient for adequate judgment. Together the experiments cover all 3 key events of skin sensitisation. The protein reactivity is addressed with the Direct Peptide Reactivity Assay (DPRA). The Keratinocyte Activation Assay LuSens assay evaluates keratinocyte activation and the Human Cell Line Activation Test (h-CLAT) determines activation of dendritic cells. An evaluation of all tests in a Weight of Evidence approach is used to reach a final conclusion on the skin sensitisation potential of the test substance and its need for classification.

DPRA:

The solubility of test item in acetonitrile at a nominal concentration of 100 mM was confirmed. Analysis of solutions of test item in the Lysine containing peptide was inconclusive due to column overload causing the Lysine peak to be unresolved and co-elute. Apart from the test item coefficient of variation in the Lysine peptide all analytical acceptance criteria were met.Therefore the DPRA prediction and the reactivity of test item was based on the mean and the individual depletion values in the cysteine peptide only.The result from the Cysteine assay places the test item in the reactivity class of “moderate reactivity” hence the test item is predicted as a potential skin sensitizer by DPRA.

LuSens

The keratinocyte activating potential of test substance was evaluated in the LuSens Assay (1928, 964.0, 482.0, 241.0, 120.5, 60.25, 30.13, 15.06, 7.53, 3.77, 1.88, and 0.94 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.

In the first experiment, a max luciferase activity (I_max) induction of 2.73 was determined at a test item concentration of 1928 µM. The corresponding cell viability was 146.8%. The lowest tested concentration with a significant luciferase induction >1.5 (1.52) was found to be 30.13 µM. The corresponding cell viability was >70% (97.4%). No EC_1.5 could be calculated.

In the second experiment, a max luciferase activity (l_max) induction of 2.26 was determined at a test item concentration of 1928 µM. The corresponding cell viability was 100.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.76) was found to be 482 µM. The corresponding cell viability was >70% (92.5%). The calculated EC_1.5 was < 1000 µM (277.16 µM).

In the third experiment, a max luciferase activity (l_max) induction of 2.16 was determined at a test item concentration of 1928 µM. The corresponding cell viability was 94.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.64) was found to be 482 µM. The corresponding cell viability was >70% (99.4%). The calculated EC_1.5 was < 1000 µM (283.63 µM).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

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

h-CLAT

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h- CLAT). The test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 274, 328, 394, 473, 567, 681, 817 and 980μg/mL.The test substance was administered to THP-1 cells for 24 ± 0.5 hours. The RFI of CD86 was greater than 150% in the two highest tested test item concentrations and the RFI of CD54 was greater than 200% in the highest tested test item concentrations in both h-CLAT runs. The two highest tested concentrations (817 and 980μg/mL) were excluded from the evaluation in both runs, as precipitations were observed. Considering this, the test item is considered to be a non-sensitiser under the test conditions of this study. The controls confirmed the validity of the study.

Conclusion

The Human Cell Line Activation Test (h- CLAT) showed no activation of dendritic cells indicating skin sensitisation potential. For protein reactivity in the Direct Peptide Reactivity Assay (DPRA) a positive result was determined which indicated a skin sensitsation potential of the test substance. As those two tests resulted in contradictory effects an additional assay addressing the third key event was conducted to reach a final conclusion. The Keratinocyte Activation Assay (LuSens) concluded a positive result for keratinocyte activation. Therefore as overall conclusion the test substance is predicted to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is considered to be classified for skin sentisation (Cat. 1b) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.