Disodium L-tyrosinate

Endpoint summary

Description of key information

Skin:

In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C (reference 7.4.1-1), the test item showed low reactivity towards cysteine or lysine, respectively under the given conditions. In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline TG 442D (reference 7.4.1 -2), the test item did not induce luciferase activity in at least two independent experimental runs. In a study according OECD TG 442E the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs (reference 7.4.1 -4). An in silico assessment of the test item did indicate a skin sensitisation potential (reference 7.4.1 -3).

Based on the results from the in chemico and in vitro studies and considering the AOP “2 out of 3” the test item in predicted as non-skin sensitizer.

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:

weight of evidence

Study period:

28 April 2017 - 18 October 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

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) - Details on study design:

Synthetic peptides used:
- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 260515HS DVWV1115
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 120514HSDW W0517
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls: cysteine or lysine peptide in acetonitrile with and without test item

Test substance preparation:
- The test substance was prepared as a 100 mM preparation in acetonitrile.

Peptide stock solution preparation:
- 19.56 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (36.19 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 20.54 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (38.982 mL) to reach a concentration of 0.667 mM.

Experimental procedure:
Three samples of the test substance in acetonitrile were incubated with each peptide for 24h at room temperature in the dark. The incubation tubes were sealed. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

Sample preparation:
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide).

HPLC conditions:
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 220 nm (Agilent, 1200 Series, with Chemstation, Rev. B.04.01) using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A (0.1% ( v/v) trifluoroacetic acid in water) and 50% phase B (0.085% ( v/v) trifluoroacetic acid in acetonitrile) for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

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.
PPD = (1- (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Reference Control C)) * 100

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.

Evaluation of results:
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.
By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89% peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

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 64.08%.

Key result

Run / experiment:

other: Cysteine

Parameter:

other: mean peptide depletion [%]

Value:

24.25

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Lysine

Parameter:

other: mean peptide depletion [%]

Value:

0.55

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 vehicle control: Yes
- Acceptance criteria met for positive control: Yes

OTHER EFFECTS:
For the cysteine and lysine peptide experiment 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.

Table 2: Depletion of the 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	1441.9841	0.1695	68.28	68.80	0.51	0.74
	1417.9863	0.1668	68.81
	1395.7390	0.1644	39.30
Test Substance	3516.7920	0.3973	20.52	24.25	3.23	13.33
	3276.0418	0.3708	25.96
	3262.4739	0.3693	26.27

Table 3: Depletion of the 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	1712.7127	0.1998	60.59	59.36	1.37	2.31
	1755.7267	0.2049	59.60
	1830.2459	0.2136	57.89
Test Substance	4276.6836	0.5001	0.57	0.55	0.08	13.84
	4280.6788	0.5005	0.47
	4274.1211	0.4998	0.63

Table 4: Categorization of the Test Item according to prediction model 1

Prediction Model	Prediction Model 1, (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)
	Peptide Depletion [%]	Reactivity Category	Prediction
Test Substance	12.4	Low Reactivity	Sensitiser
Positive Control	64.08	High Reactivity	Sensitiser

Interpretation of results:

other: peptide depletion

Remarks:

The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C, the test item showed low reactivity towards cysteine or lysine, respectively under the given conditions.

Executive summary:

In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C, the direct peptide binding potential of the test item was determined. The test substance was solved in water, based on the results of the pre-experiments. Based on a molecular weight of 225.15 g/mol a 100 mM stock solution was prepared. 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. After the 24 h ± 2 h incubation period but prior to the HPLC analysis the cysteine and lysine peptide samples were inspected for precipitation, turbidity or phase separation. For the cysteine and lysine peptide experiment 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. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria of the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant. No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C). The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was between 6.38% and 22.62% (12.40 %). Based on the prediction model 1 (Cysteine 1:10/Lysine 1:50 prediction model) the test item may be considered as sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 May 2017 - 22 September 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

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SLBR2521V
- Expiration date of the lot/batch: May 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 °C, protected from light

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.

Cell line used: KeratinoSensTM (Givaudan, Switzerland)

Technical material and conditions:
- Maintenance Medium: D-MEM (GlutaMAXTM) with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS + 1 % Geneticin (final concentration: 500 µg/mL)
- Assay Medium: D-MEM (GlutaMAXTM) 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 in DPBS
- SDS solution: 10% (w/v) SDS in dist. water
- DPBS solution: DPBS solution (without Ca2+/Mg2+)

Controls used:
- Vehicle control: DMSO: 1% (v/v) 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 item: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.

Test procedure:
Each concentration step of the test item (twelve concentrations, 0.98 - 2000 µM) and the positive control (5 concentrations, 4 - 64 µM) was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run. 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. The OD was measured at a wavelength of 600 nm.

Data analysis:
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consists of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run should be performed.

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 luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (5.70 in experiment 1; 6.31 in experiment 2; 2.27 in experiment 3).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity; Imax

Value:

2.46

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

100.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Value:

621.42

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity, Imax

Value:

1.55

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

78.2

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

1 884.19

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: Luciferase activity, Imax

Value:

1.16

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: Cell viability [%]

Value:

86.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: 3

Parameter:

other: EC 1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Additional Parameters

Parameter	Experiment 1	Experiment 2	Experiment 3	Mean	SD
EC1.5[µM]	621.42	1884.19	n.a.	1262.81	892.91
Imax	2.46	1.55	1.16	1.72	0.67
IC30[µM]	n.a.	n.a.	n.a.	-	-
IC50[µM]	n.a.	n.a.	n.a.	-	-

n.a. = not applicable

Table 2: Results of the Cytotoxicity Measurement

	Concentration	Cell Viability [%]
	[µM]	Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	100	100	100	100	0.0
Positive Control	4.00	102.4	94.3	104.5	100.4	5.4
	8.00 16.00	105.5 112.2	102.2 102.4	108.8 112.4	105.5 109.0	3.3 5.7
	32.00	106.2	105.8	119.4	110.5	7.7
	64.00	121.1	106.7	98.0	108.6	11.7
Test Item	0.98	102.3	100.7	96.5	99.8	3.0
	1.95	101.2	94.9	95.4	97.2	3.5
	3.91	100.8	94.3	94.7	96.6	3.6
	7.81	98.3	96.0	94.0	96.1	2.2
	15.63	104.9	94.5	90.1	96.5	7.6
	31.25 62.50	97.8 92.3	78.8 90.7	93.2 93.3	89.9 92.1	9.9 1.3
	125.00	91.6	90.3	97.9	93.2	4.1
	250.00	95.8	96.4	94.1	95.5	1.2
	500.00	91.7	93.2	96.6	93.8	2.5
	1000.00	101.4	90.2	91.1	94.2	6.3
	2000.00	100.4	78.2	86.4	88.3	11.2

 

Table 3: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration	Fold Induction					Significance
	[µM]	Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.17	1.33	1.21	1.24	0.08
	8.00 16.00 32.00	1.35 1.46 2.11	1.34 1.55 1.98	1.08 1.27 1.64	1.25 1.43 1.91	0.15 0.14 0.24	*
	64.00	6.96	5.89	4.26	5.70	1.36	*
Test Item	0.98	1.58	1.55	1.57	1.57	0.02
	1.95	1.54	1.44	1.29	1.42	0.13
	3.91	1.63	1.71	1.48	1.61	0.11	*
	7.81	1.65	1.71	1.42	1.60	0.15	*
	15.63	1.79	1.79	1.47	1.68	0.19	*
	31.25 62.50	1.72 1.17	1.91 1.23	1.49 0.99	1.71 1.13	0.21 0.12	*
	125.00	1.60	1.67	1.33	1.53	0.18	*
	250.00	1.07	1.22	0.97	1.09	0.13
	500.00	1.38	1.49	1.08	1.32	0.21
	1000.00	2.13	2.30	1.79	2.07	0.26	*
	2000.00	2.63	2.66	2.07	2.46	0.33	*

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

 

Table 4: 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	1.19	1.24	1.06	1.16	0.09
	8.00	1.16	1.12	1.32	1.20	0.11
	16.00	1.37	1.55	1.59	1.50	0.12	*
	32.00	2.02	2.27	2.16	2.15	0.13	*
	64.00	6.51	5.74	6.68	6.31	0.50	*
Test Item	0.98	1.04	1.10	0.95	1.03	0.07
	1.95	0.90	0.90	1.01	0.93	0.06
	3.91	0.78	1.12	0.90	0.93	0.17
	7.81	0.93	0.82	0.96	0.91	0.08
	15.63	0.96	0.88	0.82	0.89	0.07
	31.25	0.90	0.77	1.03	0.90	0.13
	62.50	0.92	0.84	0.89	0.88	0.04
	125.00	0.96	0.88	1.01	0.95	0.07
	250.00	0.95	0.95	0.98	0.96	0.02
	500.00	0.94	0.84	1.13	0.97	0.15
	1000.00	1.14	1.08	1.17	1.13	0.05
	2000.00	1.89	1.29	1.47	1.55	0.31

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

 

Table 5: Induction of Luciferase Activity Experiment 3

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	1.06	1.08	1.12	1.09	0.03
	8.00	1.11	1.28	1.34	1.24	0.12
	16.00	1.27	1.31	1.43	1.33	0.08
	32.00	1.52	1.47	2.43	1.81	0.54
	64.00	2.17	2.30	2.33	2.27	0.09	*
Test Item	0.98	1.14	1.16	1.09	1.13	0.04
	1.95	0.95	1.21	1.04	1.07	0.13
	3.91	0.93	1.08	1.05	1.02	0.08
	7.81	0.87	1.05	0.99	0.97	0.09
	15.63	0.95	1.19	1.03	1.06	0.13
	31.25	1.00	0.97	0.95	0.97	0.03
	62.50	0.97	1.05	1.00	1.00	0.04
	125.00	0.89	1.02	1.09	1.00	0.10
	250.00	0.99	0.99	1.07	1.01	0.04
	500.00	0.90	0.98	0.99	0.96	0.05
	1000.00	0.83	1.03	1.27	1.05	0.22
	2000.00	1.06	1.20	1.22	1.16	0.08

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

 

Table 5: Induction of Luciferase Activity - Overall Induction

	Concentration [µM]	Fold Induction					Significance
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.24	1.16	1.09	1.16	0.08
	8.00 16.00 32.00	1.25 1.43 1.91	1.20 1.50 2.15	1.24 1.33 1.81	1.23 1.42 1.96	0.03 0.08 0.18	*
	64.00	5.70	6.31	2.27	4.76	2.18	*
Test Item	0.98	1.57	1.03	1.13	1.24	0.29
	1.95	1.42	0.93	1.07	1.14	0.25
	3.91	1.61	0.93	1.02	1.19	0.37
	7.81	1.60	0.91	0.97	1.16	0.38
	15.63	1.68	0.89	1.06	1.21	0.42
	31.25	1.71	0.90	0.97	1.19	0.44
	62.50	1.13	0.88	1.00	1.01	0.12
	125.00	1.53	0.95	1.00	1.16	0.32
	250.00	1.09	0.96	1.01	1.02	0.06
	500.00	1.32	0.97	0.96	1.08	0.20
	1000.00	2.07	1.13	1.05	1.42	0.57
	2000.00	2.46	1.55	1.16	1.72	0.67

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

 

Table 6: Acceptance Criteria

Criterion	Range	Exp. 1	pass/fail	Exp. 2	pass/fail	Exp. 3	pass/fail
CV Solvent Control	< 20%	9.9	pass	8.4	pass	6.4	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	> 1	2.0	pass	3.0	pass	1.0	pass
EC1.5 PC	7 < x < 34 µM	18.43	pass	15.90	pass	21.59	pass
Induction PC at 64 µM	2.00 < x < 8.00	5.70	pass	6.31	pass	2.27	pass

Table 8: 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

 

Interpretation of results:

other: no activation of keratinocytes

Remarks:

The 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 integrated approach such as IATA.

Conclusions:

In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline 442D, the test item did not induce luciferase activity.

Executive summary:

In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD TG 442D, the skin sensitising potential of the test item was determined. The test item was dissolved in distilled water. Based on a molecular weight of 225.15 g/mol a stock solution of 200 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:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 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 2.46 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 100.4%. The lowest tested concentration with a significant luciferase induction > 1.5 (2.07) was found to be 1000 µM. The corresponding cell viability was > 70% (101.4%). The calculated EC1.5 was < 1000 µM (621.42 µM).

In the second experiment, a max. luciferase activity (I_max) induction of 1.55 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 78.2%. No further significant luciferase induction > 1.5 was found in the tested concentration range. The calculated EC1.5 was > 1000 µM (1884.19 µM).

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

A slight dose response for luciferase activity induction was observed for experiment 1 and experiment 2, whereas no dose response for luciferase activity induction was observed within the third experiment.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-04 to 2018-03-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)”

Version / remarks:

29 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

Version / remarks:

July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SLBR2521V

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 °C, protected from light

Details on the study design:

Skin sensitisation (In vitro test system) - Details on 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.

Preparation of the Test Item
The test item was freshly prepared immediately prior to use. The test item was dissolved in 0.9% NaCl solution at a concentration of 500 mg/mL. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 50 times with cell culture medium. No precipitation or phase separation was observed when diluted 1:50 in cell culture medium. A colour change of the medium to pink was observed for the first three concentration steps of the test item in the main experiment. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (0.9% NaCl solution) was present at a constant volume ratio of 1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Dose Finding Assay
Starting from 100 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 50-fold into culture medium (working solutions). Since 0.9% NaCl or medium was used as solvent and the top concentration of 1000 μg/mL was non-toxic, the maximum concentration was re-determined by performing a new cytotoxicity test, up to a final concentration of 5000 μg/mL. The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution. For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10E6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10E6 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10E6 cells/well). The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 μL FACS buffer. 200 μL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 μg/mL. The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration according to equation. The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation.

Dose groups
8 concentrations of the test item
dose finding assay 1:
1000.00, 500.00, 250.00, 125.00, 62.50, 31.25, 15.63 and 7.81 μg/mL
dose finding assay 2:
5000.00, 2500.00, 1250.00, 625.00, 312.50, 156.25, 78.13, 39.06 μg/mL
main experiment 1 and 2:
5000.00, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49, 1395.41 μg/mL

Controls
Medium Control: A medium control was included in the test.
Solvent Controls: Solvent controls were included in the test. The solvent controls were set up depending on the appropriate solvent previously determined.Since the test item was solubilized in either cell culture medium or 0.9% NaCl, the medium control served as solvent control. Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the positive control. The solvent controls were diluted according to the procedure described for the test item, resulting in a final concentration of 1% (v/v) for 0.9% NaCl and 0.2% (v/v) for DMSO.
Positive Control: 2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described for the test item in, resulting in a final DMSO concentration of 0.2% (v/v).

Cell line
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10E6 cells/mL. Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 μg/mL streptomycin at 37 ± 1°C and 5% CO2.

CD54 and CD86 Expression
The test item was dissolved using 0.9% NaCl as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 100-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 50-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor. For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 10E6 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10E6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 μL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 μg/mL). The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was calculated.

Antibodies
FITC-labelled anti-CD86: BD, art. no. 555657, Lot No.: 7132528
FITC-labelled anti-CD54: BioLegend, art. no.: 353108, Lot No.:B214036
FITC-labelled mouse IgG1 (isotype): BioLegend, art. no.: 400110, Lot No.:B206037

Data Analysis
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 and Effective Concentration 200 values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.

Prediction Model
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 μg/mL for 0.9% NaCl solution; 1000 μg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.

Effective Concentration 150 and Effective Concentration 200 values:
For test chemicals classified as sensitiser the effective concentration 150 for CD86 (EC150) and the effective concentration 200 for CD54 (EC200) can be calculated. If the RFI value of the lowest dose is above the positive criteria of CD86 and CD54, in conformity with DB-ALM Protocol n°158 the EC150 and EC200 values can be calculated using the lowest dose by log linear extrapolation.

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%.

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 (333% experiment 1; 408% experiment 2) and 200% for CD54 (373% experiment 1; 442% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

128

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 5000 µM

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 2893.52 µM

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

127

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 3472.22 µM

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

116

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 3472.22 µM

Other effects / acceptance of results:

OTHER EFFECTS:
Doubling time of the cells was monitored and found to be 38.6 h (Batch 18) and 44.6 h (Batch 19) which is within the doubling time range specified by the manufacturer (35 - 50 h).


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Results of the Dose Finding Assay

Sample	Experiment 1		Experiment 2
	Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	96.80	--	94.70
Solvent Control (NaCl)	--	96.80	--	94.60
test item	7.81	96.90	39.06	96.10
	15.63	96.90	78.13	95.50
	31.25	95.70	156.25	94.30
	62.50	96.20	312.50	94.90
	125.00	95.90	625.00	96.10
	250.00	96.50	1250.00	95.40
	500.00	96.60	2500.00	93.90
	1000.00	96.00	5000.00	80.90
Calculated CV75 [µg/mL]	No CV75		No CV75
Mean CV75 [µg/mL]	No CV75
SD CV 75 [µg/mL]	No SD

Table 2: 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	-	97.3	97.6	96.6	2232	1078	617	1615	461	100	100	362	175
Solvent Control (DMSO)	0.20%	96.6	97.0	96.6	2609	1107	651	1958	456	121	99	401	170
DNCB	4.00	87.7	86.2	85.9	7347	2521	822	6525	1699	333	373	894	307
test item	5000	96.1	96.3	96.4	2678	1024	608	2070	416	128	90	440	168
	4166.67	96.7	97.1	96.4	2667	1050	715	1952	335	121	73	373	147
	3472.22	97.1	96.2	96.6	2456	1069	656	1800	413	111	90	374	163
	2893.52	96.2	96.4	95.7	2529	1079	618	1911	461	118	100	409	175
	2411.27	96.9	96.5	97.1	2479	1014	604	1875	410	116	89	410	168
	2009.39	96.9	96.9	96.7	2365	1049	706	1659	343	103	74	335	149
	1674.49	96.9	96.6	96.6	2379	1024	651	1728	373	107	81	365	157
	1395.41	96.8	96.5	96.4	2319	1064	617	1702	447	105	97	376	172

Table 3: 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	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	96.7	96.6	96.1	2079	1076	555	1524	521	100	100	375	194
Solvent Control (DMSO)	0.20%	96.3	96.5	95.9	2044	1111	585	1459	526	96	101	349	190
DNCB	4.0	83.6	83.6	83.0	6817	3189	863	5954	2326	408	442	790	370
test item	5000.00	95.5	95.8	95.6	2350	1064	612	1738	452	114	87	384	174
	4166.67	96.3	96.1	96.0	2507	1123	701	1806	422	119	81	358	160
	3472.22	96.3	96.2	96.5	2546	1219	617	1929	602	127	116	413	198
	2893.52	96.3	96.0	96.0	2488	1100	677	1811	423	119	81	368	162
	2411.27	96.1	96.4	96.2	2324	1077	605	1719	472	113	91	384	178
	2009.39	96.4	95.8	96.2	2580	1159	700	1880	459	123	88	369	166
	1674.49	96.5	95.9	96.4	2348	1095	602	1746	493	115	95	390	182
	1395.41	96.2	96.3	95.9	2387	1104	606	1781	498	117	96	394	182

Table 4: Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	96.6	-	97.6	pass	95.9	-	96.7	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	333			pass	408			pass
RFI of positive control of CD54	≥200	373			pass	442			pass
RFI of solvent control of CD86	<150	121			pass	96			pass
RFI of solvent control of CD54	<200	99			pass	101			pass
MFI ratio IgG1/CD86 for medium control [%]	>105	362			pass	375			pass
MFI ratio IgG1/CD86 for DMSO control [%]	>105	401			pass	349			pass
MFI ratio IgG1/CD54 for medium control [%]	>105	175			pass	194			pass
MFI ratio IgG1/CD54 for DMSO control [%]	>105	170			pass	190			pass

Table 5: 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
MFI ratio IgG1/CD54 for DMSO control [%]	147.7	25.6	112

Interpretation of results:

other: no activation of dendritic cells

Remarks:

The 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 integrated approach such as IATA.

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs.

Executive summary:

To assess the skin sensitising potential of the test item a GLP-compliant in vitro human cell line activation test (h-CLAT) according to OECD TG 442E was carried out using THP-1 cells (ATCC® TIB-202TM). 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. The test item was dissolved in 0.9% NaCl. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore the main experiment was performed covering the following concentration steps: 5000; 4166.67; 3472.22; 2411.27; 2009.39; 1674.49; 1395.41 µg/mL. A medium control (cell culture medium only), a solvent control (cell culture medium only), and a positive control (with 2,4-dinitrochlorobenzene) were set up in parallel in order to confirm the validity of the test. 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. In result, 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 96.1% (CD86), 96.3% (CD54) and 96.4% (isotype IgG1 control) in the first experiment and to 95.5% (CD86), 95.8% (CD54) and 95.6% (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. The positive control led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (333% experiment 1; 408% experiment 2) and 200% for CD54 (373% experiment 1; 442% experiment 2) were clearly exceeded. All acceptance criteria of the guideline were met. In conclusion, the test item did not upregulate the expression of the cell surface marker.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

Please refer to the QMRF and QPRF files provided under the section attached justification.

Qualifier:

no guideline available

Principles of method if other than guideline:

Estimates the skin sensitising properties of chemicals using structural alert relationships.

GLP compliance:

no

Specific details on test material used for the study:

SMILES: [Na+].[Na+].N[C@@H](Cc1ccc(O)cc1)C(=O)[O-].N[C@@H](Cc2ccc(O)cc2)C(=O)[O-]

Key result

Parameter:

other: alerts

Value:

1

Remarks on result:

other: equivocal

Remarks:

QSAR predicted value. The substance is within the applicability domain of the model.

Interpretation of results:

other: Derek result: Alert 439 has been detected.

Conclusions:

Using Derek Nexus v5.0, skin sensitising properties of the test item were regarded as equivocal. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

Executive summary:

The skin sensitising properties were estimated using Derek Nexus v5.0. Skin sensitising properties were regarded as equivocal based on the described QSAR method (Derek, 2017).

Alert 439 (Substituted phenol or precursor) was detected for the test substance. The alert has demonstrated the following predictive performance:

1) Cronin and Basketter: 18 compounds activate this alert of which 4 are reported positive. (Positive predictivity: 22%.)

2) Gerberick: 18 compounds activate this alert of which 8 are reported positive. (Positive predictivity: 44%.)

3) Contact Dermatitis: 5 compounds activate this alert of which 2 are reported positive. (Positive predictivity: 40%.)

In assessing predictive performance, it should be noted that:

- Mammalian skin sensitisation predictions in Derek associated with a reasoning level of equivocal or above have been considered positive;

- Predictions do not take into account (i) the tautomeric forms of compounds or (ii) the individual components of mixtures;

- Compounds have been considered positive for skin sensitisation if they have been classified as extreme, strong or moderate sensitisers;

- Compounds classified as weak sensitisers have been excluded from the analysis;

- No account has been taken of other skin sensitisation alerts which may also be present in some compounds;

- Information from the data sets may have been used previously as supporting evidence for the derivation of some alerts;

- Some compounds may be present in more than one of the data sets analysed.

Although phenol itself gave negative results in animal tests for skin sensitisation [Itoh, Basketter et al 1999], positive results have been reported for several substituted derivatives, including O-methyl precursors. Examples of compounds identified as skin sensitisers in the LLNA include 2,5-dimethyl phenol, 3,4-dimethyl phenol [Yamano et al], pentachlorophenol [Basketter et al 1996], 2,4,5-trichlorophenol [Ashby et al] and 1-methoxy-4-propenylbenzene (trans-anethol) [Gerberick et al]. Positive results have also been reported for several substituted phenols and precursors in the GPMT. Examples include 4-styrylphenol (4-hydroxystilbene), 1-methoxy-4-propenylbenzene (anethole), 4- allylanisole [Cronin and Basketter] and chloroxylenol (4-chloro-2,3-dimethylphenol) [Goh]. It is important to note that negative results have also been reported for several substituted phenols and precursors in both the GPMT and the LLNA. Compounds reported as negative in the GPMT or the LLNA include 4-tert-butylanisole [Cronin and Basketter], 4-tert-butylphenol [Zimerson et al], 2-methylphenol and 3,4,5-trimethylphenol [Yamano et al].

Substituted phenols have given mixed results in the human maximisation test. For example, while negative results were reported for p-cresol [Opdyke 1974] and hexachlorophene [Kligman], positive results have been reported for alpha-methylanisalacetone [Opdyke 1979] and pentachlorophenol [Basketter et al 1996]. Diagnostic human patch tests have identified several substituted phenols, including 2-methylolphenol, 2,4-methylolphenol and o-cresol, as contact sensitisers to patients hypersensitive to phenol formaldehyde resin [Bruze and Zimerson].

There are multiple mechanisms by which substituted phenols may induce skin sensitisation. These include: (i) Abstraction of the hydroxyl hydrogen atom to generate a reactive phenolic radical species. Subsequent reaction with skin proteins may occur directly or via hydrogen abstraction from a protein thiol group. The resulting thiyl radical may then react with a second phenolic radical species [Barratt and Basketter]. (ii) Ring oxidation to yield the corresponding catechol or hydroquinone. Further enzymatic or auto-oxidation leads to the formation of electrophilic ortho- or para-quinones capable of reacting directly with skin proteins via Michael addition [Barratt and Basketter]. (iii) For some ortho- or para-alkyl substituted phenols, skin sensitisation may be induced via formation of corresponding ortho- or para-quinone methides. These species may also be able to react with skin proteins via Michael addition [Barratt and Basketter].

Substituted phenols and precursors may induce skin sensitisation via multiple potentially overlapping mechanisms and the toxicity data for these compounds are mixed. As a result, the scope of this alert covers a wide range of structurally diverse compounds. Hydrogen, alkyl, aryl and halogen substituents are all permitted on the phenol ring as are additional aromatic ring fusions. This is based on the activity of compounds with each of these structural features in animal tests for skin sensitisation. O-Methyl precursors are included based on the activity of several anisole compounds and an expectation that the methyl group will be cleaved enzymatically in the skin [Barratt and Basketter]. At least one non-hydrogen substituent is required based on the inactivity of phenol in the GPMT and the LLNA. Carbon substituents are permitted in both ortho-positions, however at least one substituent must have one or more hydrogen atoms attached. This is based on the inactivity of several 2,6-di-tert-butylphenols, including 2,6-di-tert-butylphenol [ECHA], in the GPMT.

Oestradiol-type steroidal structures lacking substitution on, and carbon double bond conjugation of, the A ring are excluded from the alert. This is based on limited proprietary data provided by Bayer Schering Pharma AG indicating no evidence of skin sensitisation potential for compounds of this type in animal experiments. In addition, oestradiol itself is in wide-spread human use in transdermally administered hormone replacement therapy. Although isolated case reports have described allergic contact dermatitis to oestradiol (or in some cases are suggestive of it) following use of hormone replacement therapy [Boehncke and Gall, Carmichael and Foulds, Goncalo et al, Quirce et al], clinical trial data indicate that in general dermal exposure to oestradiol under such conditions has no sensitising potential [Rozenbaum et al].

For further details also on the References, please refer to the attached QPRF document.

The adequacy of above prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model isapplicable to thequery chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model isrelevant for theregulatory purpose.

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. The protein reactivity of the test item was assessed in a Direct Peptide Reactivity Assay (DPRA). A Keratinocyte Activation Assay (KeratinoSens) was performed to evaluate the keratinocyte activation potential. To assess events in dendritic cells a Human Cell Line Activation Test (h-CLAT) was performed. Furthermore, an in silico assessment using Derek Nexus v5.0 was carried out.

DPRA (reference 7.4.1 -1)

The direct peptide reactivity assay (DPRA) addresses the first key event in skin sensitisation, according to the defined adverse outcome pathway (AOP). Therefore, the direct peptide binding potential of the test item was determined in an in chemico DPRA according to OECD Guideline 442C (reference 7.4.1-1), The test item was solved in water, based on the results of the pre-experiments. Based on a molecular weight of 225.15 g/mol a 100 mM stock solution was prepared. The test substance 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. After the 24 h ± 2 h incubation period but prior to the HPLC analysis the cysteine and lysine peptide samples were inspected for precipitation, turbidity or phase separation. For the cysteine peptide experiment 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. Samples were not centrifuged prior to the HPLC analysis. For the lysine peptide experiment no precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria of the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant. No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C. The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (12.40%). Based on the prediction model 1 the test item may be considered as sensitiser.

KeratinoSens (reference 7.4.1-2)

The KeratinoSens™ assay is supposed to address the second key event of the skin sensitisation process as defined by the AOP, the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress.

Accordingly, the skin sensitising potential of the test item was determined in an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline 442D, The test item was dissolved in distilled water. Based on a molecular weight of 225.15 g/mol a stock solution of 200 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 concentrations were tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 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 2.46 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 100.4%. The lowest tested concentration with a significant luciferase induction >1.5 (2.07) was found to be 1000 µM. The corresponding cell viability was >70% (101.4%). The calculated EC1.5 was < 1000 µM (621.42 µM).

In the second experiment, a max luciferase activity (I_max) induction of 1.55 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 78.2%. No further significant luciferase induction >1.5 was found in the tested concentration range. The calculated EC1.5 was > 1000 µM (1884.19 µM). In the third experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. A slight dose response for luciferase activity induction was observed for experiment 1 and experiment 2, whereas no dose response for luciferase activity induction was observed within the third experiment.

Human cell line activation test (hCLAT) (reference 7.4.1-4)

To assess the skin sensitising potential of the test item a GLP-compliant in vitro human cell line activation test (h-CLAT) according to OECD TG 442E was carried out using THP-1 cells (ATCC® TIB-202TM). 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. The test item was dissolved in 0.9% NaCl. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore the main experiment was performed covering the following concentration steps: 5000; 4166.67; 3472.22; 2411.27; 2009.39; 1674.49; 1395.41 µg/mL. A medium control (cell culture medium only), a solvent control (cell culture medium only), and a positive control (with2,4-dinitrochlorobenzene) were set up in parallel in order to confirm the validity of the test. 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. In result, 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 96.1% (CD86), 96.3% (CD54) and 96.4% (isotype IgG1 control) in the first experiment and to 95.5% (CD86), 95.8% (CD54) and 95.6% (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. The positive control led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (333% experiment 1; 408% experiment 2) and 200% for CD54 (373% experiment 1; 442% experiment 2) were clearly exceeded. All acceptance criteria of the guideline were met. In conclusion, the test item did not upregulate the expression of the cell surface marker.

In silico assessment (reference 7.4.1-3)

For in silico assessment, Derek Nexus v5.0 was used. Skin sensitising properties were regarded as equivocal based on the described QSAR method (Derek, 2017). Alert 439 (Substituted phenol or precursor) was detected for the test substance. In consequence, the test item is suspected to contain skin sensitising properties.

Conclusion:

The first two assays (OECD TG 442C and OECD TG 442D) provided discordant results. Therefore, an assay for the third key event (activation of dendritic cells) was performed. The overall result is based on two concordant findings. Considering an AOP “2 out of 3” the test item in predicted as non-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 (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is not classified as skin sensitising according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.