2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(1H-imidazol-5-yl)propanoyl]amino]-2-methyl-propanoic acid Trifluoroacetic acid salt (1:1)

Endpoint summary

Description of key information

OECD 442D:

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

OECD 442E:2018:

Under the test conditions, the test substance resulted to be NEGATIVE (NON-SENSITIZER) up to the maximal tested concentration of 500μg/mL.

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:

Study Initiation Date: 24 June 2020 - Study Completion Date: 22 December 2020

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 KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

Name: FMOC-HIS-AIB-OH TFA LS
Chemical Name: 2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-(1H-imidazol-5-yl)propanoyl]amino]-2-methyl-propanoic acid Trifluoroacetic acid s
Batch No.: 0020000582
CAS No.: 1446013-08-6
Molecular Weight: 576.51 g/mol
Purity (HPLC): 99.7%
Physical State: powder
Colour: white
Stability: stable for 3 years
Storage Conditions: 5 ± 3°C (Fridge)
Expiry Date: 16 May 2023
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D
- Preparation of the Test Item
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%). A stock solution of 200 mM was prepared by pre-weighing the test material into a suitable tube. Vortex mixing was used to aid solubilisation.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
- Controls
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.
Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
Negative Control
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.
- Cell line
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 04 in experiment 1; P 06 in experiment 2 and P 08 in experiment 3) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37  1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.
- Composition of Media
Maintenance Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
10% fetal bovine calf serum (Biochrom, Cat. No.: S 0615)
1% geneticin (final concentration: 500 µg/mL; Gibco Life Science, Cat. No. 2009535)
Assay Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
10% fetal bovine calf serum (Biochrom, Cat. No.: S 0615)
Test Item Exposure Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with the following components:
1% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 1000F)

- Luciferase Assay System
Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501) consisted of the following components relevant for this study:
10 vials Luciferase Assay Substrate (lyophilized)
10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer.
If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531) consisted of the following components relevant for this study:
30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma)
- Further Reagents
MTT solution
MTT (VWR, CAS No.: 298-93-1) stock solution: 5 mg/mL MTT in DPBS (Gibco Life Science)
SDS solution:
10% (w/v) sodium dodecyl sulfate (SDS; AppliChem, CAS No.: 151-21-3) in dist. water (Sigma)
DPBS:
DPBS solution (without Ca2+/Mg2+) (Gibco Life Science)
- Dose Groups
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
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 96-well plate in every independent run.
 
- Experimental Procedure
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.
Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2) or over the weekend (experiment 3). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 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 consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed.
The following parameters were calculated:

Calculation of Cell Viability
Cell viability was calculated according to equation 1.
Cell Viability [%]= ((V_sample-V_blank))/((V_solvent-V_blank))×100 Equation 1
Vsample = MTT absorbance reading in the test chemical well
Vblank = MTT absorbance reading in the blank well containing no cells and no treatment
Vsolvent = average MTT absorbance reading in the wells containing cells and solvent (negative) control

Calculation of the Maximal Induction of the Luciferase Activity (Imax)
The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item was calculated according to equation 2.
Fold Induction = ((L_sample-L_blank))/((L_solvent-L_blank)) Equation 2
Lsample = luminescence reading in the test chemical well
Lblank = luminescence reading in the blank well containing no cells and no treatment
Lsolvent = luminescence reading in the wells containing cells and solvent (negative) control

Calculation of the EC1.5
The EC1.5 will be calculated by linear extrapolation according to equation 3, and the overall EC1.5 was calculated as the geometric mean of the individual repetitions.
EC1.5=(Cb-Ca )×((1.5-Ia)/(Ib-Ia ))+Ca Equation 3
Ca = lowest concentration in µM with >1.5 fold induction
Cb = highest concentration in µM with <1.5 fold induction
Ia = fold-induction measured at the lowest concentration with >1.5 fold induction (mean of three replicate wells)
Ib = fold-induction measured at the highest concentration with <1.5 fold induction (mean of three replicate wells)

Calculation of IC50 and IC30
The IC50 and IC30 were calculated by linear extrapolation according to equation 4 and the overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.
ICx=(Cb-Ca )×(((100-x)-Va))/(Vb-Va ))+Ca Equation 4
X = % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca = the lowest concentration in µM with >X% reduction in cell viability
Cb = highest concentration in µM with Va = % viability at the lowest concentration with >X% reduction in cell viability
Vb = % viability at the highest concentration with

For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.

The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.
- Prediction Model
A KeratinoSens™ prediction is considered positive if the following conditions will be 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. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.

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

Vehicle / solvent control:

DMSO

Negative control:

other: DMSOat a final concentration of 1% (v/v) in test item exposure medium

Positive control:

cinnamic aldehyde [442D]

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

9.89 µM

Cell viability:

The max luciferase activity (Imax) induction of 1.92 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 92.3%.
The lowest tested concentration with a significant luciferase induction >1.5 (1.88) was found to be 15.63 µM. The corresponding cell viability was >70% (89.1%).

Vehicle controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

In the first experiment, no dose response for luciferase activity induction was observed and the test item is therefore considered as inconclusive.

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Remarks on result:

not determinable

Remarks:

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

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Remarks on result:

not determinable

Remarks:

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

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

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.
In the present study FMOC-HIS-AIB-OH TFA LS was dissolved in DMSO. Based on a molecular weight of 576.51 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 (Imax) induction of 1.92 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 92.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.88) was found to be 15.63 µM. The corresponding cell viability was >70% (89.1%). The calculated EC1.5 was <1000 µM (9.89 µM).
In the first experiment, no dose response for luciferase activity induction was observed and the test item is therefore considered as inconclusive.
In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. Therefore, the test item can be considered as non-sensitiser in the second experiment.
With two different outcomes the guideline OECD442D recommends to do a third experiment for classification.
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.
No dose response for luciferase activity induction was observed for two independent runs as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non-sensitiser.
The controls confirmed the validity of the study (see Table 7)

Results

 Cytotoxicity

Table1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Experiment 3	Mean	SD
Solvent Control	-	100	100	100	100	0.0
Positive Control	4.00	144.6	99.4	100.7	114.9	25.7
	8.00	124.7	97.1	102.0	108.0	14.7
	16.00	139.4	109.4	106.2	118.4	18.3
	32.00	123.0	113.2	111.6	115.9	6.1
	64.00	72.8	106.0	108.5	95.8	19.9
Test Item	0.98	76.1	77.4	88.5	80.7	6.8
	1.95	89.3	87.8	98.0	91.7	5.5
	3.91	88.6	90.4	94.9	91.3	3.3
	7.81	80.8	84.8	92.7	86.1	6.1
	15.63	89.1	91.8	92.9	91.3	2.0
	31.25	92.3	89.5	92.9	91.6	1.8
	62.50	111.3	90.8	92.3	98.1	11.4
	125.00	98.6	88.8	88.2	91.9	5.8
	250.00	88.4	90.1	87.3	88.6	1.4
	500.00	79.7	92.4	88.6	86.9	6.5
	1000.00	0.1	0.3	81.3	27.2	46.8
	2000.00	0.1	0.7	82.0	27.6	47.1

 

Luciferase Activity Experiment 1

Table2: 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	1.08	0.95	0.90	0.98	0.09
	8.00	1.28	1.31	1.03	1.21	0.15
	16.00	1.72	1.48	1.27	1.49	0.23
	32.00	1.91	1.77	1.61	1.76	0.15	*
	64.00	3.11	3.69	3.00	3.27	0.37	*
Test Item	0.98	1.08	1.44	1.47	1.33	0.22
	1.95	1.40	1.18	1.41	1.33	0.13
	3.91	1.22	1.44	1.25	1.30	0.12
	7.81	1.63	1.34	1.12	1.36	0.26
	15.63	1.86	2.42	1.35	1.88	0.54	*
	31.25	2.06	2.58	1.12	1.92	0.74
	62.50	2.56	1.01	1.09	1.56	0.87
	125.00	2.27	1.15	1.24	1.55	0.62
	250.00	2.96	1.02	1.09	1.69	1.10
	500.00	0.00	0.01	0.00	0.01	0.01
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

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

 

Luciferase Activity Experiment 2

Table3: 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	1.11	1.02	1.17	1.10	0.08
	8.00	1.17	1.15	1.25	1.19	0.05
	16.00	1.45	1.05	1.12	1.20	0.22
	32.00	1.70	1.54	1.62	1.62	0.08	*
	64.00	2.61	2.18	2.78	2.52	0.31	*
Test Item	0.98	1.09	1.53	1.35	1.32	0.22
	1.95	0.92	1.15	1.02	1.03	0.12
	3.91	1.02	1.08	0.99	1.03	0.05
	7.81	0.91	0.99	0.97	0.96	0.04
	15.63	0.87	1.04	0.98	0.96	0.08
	31.25	0.94	1.15	0.99	1.03	0.11
	62.50	0.85	1.09	0.99	0.98	0.12
	125.00	0.89	1.04	1.02	0.98	0.08
	250.00	1.06	1.10	0.98	1.05	0.06
	500.00	1.03	1.19	1.11	1.11	0.08
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

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

 

Luciferase Activity Experiment 3

Table4: 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	0.92	1.15	1.10	1.06	0.12
	8.00	1.04	1.27	1.04	1.11	0.13
	16.00	1.27	1.43	1.34	1.35	0.08
	32.00	1.91	2.04	1.91	1.95	0.07	*
	64.00	2.54	3.70	3.21	3.15	0.58	*
Test Item	0.98	1.28	1.77	0.74	1.27	0.51
	1.95	1.11	1.28	0.59	1.00	0.36
	3.91	1.05	1.40	0.63	1.03	0.39
	7.81	0.96	1.44	0.67	1.02	0.39
	15.63	0.97	1.30	0.71	0.99	0.29
	31.25	1.02	1.15	0.69	0.95	0.23
	62.50	1.09	1.22	0.92	1.07	0.15
	125.00	1.13	1.16	0.85	1.05	0.17
	250.00	1.19	1.30	0.83	1.10	0.24
	500.00	1.16	1.22	0.99	1.12	0.12
	1000.00	0.08	0.00	0.00	0.03	0.05
	2000.00	0.99	1.50	0.64	1.04	0.43

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

Luciferase Activity - Overall Induction

Table 5: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction					Significance
		Exp. 1	Exp. 2	Exp. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	0.98	1.10	1.06	1.05	0.06
	8.00	1.21	1.19	1.11	1.17	0.05
	16.00	1.49	1.20	1.35	1.35	0.14
	32.00	1.76	1.62	1.95	1.78	0.17	*
	64.00	3.27	2.52	3.15	2.98	0.40	*
Test Item	0.98	1.33	1.32	1.27	1.30	0.03
	1.95	1.33	1.03	1.00	1.12	0.18
	3.91	1.30	1.03	1.03	1.12	0.16
	7.81	1.36	0.96	1.02	1.12	0.22
	15.63	1.88	0.96	0.99	1.28	0.52
	31.25	1.92	1.03	0.95	1.30	0.54
	62.50	1.56	0.98	1.07	1.20	0.31
	125.00	1.55	0.98	1.05	1.19	0.31
	250.00	1.69	1.05	1.10	1.28	0.36
	500.00	0.01	1.11	1.12	0.75	0.64
	1000.00	0.00	0.00	0.03	0.01	0.02
	2000.00	0.00	0.00	1.04	0.35	0.60

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

 Additional Parameters

Table6: Additional Parameters

Parameter	Experiment 1	Experiment 2	Experiment 2	Mean	SD
EC1.5[µM]	9.89	n.a.	n.a.	n.a.	n.a.
Imax	1.92	1.32	1.27	1.50	0.36
IC30[µM]	561.13	621.61	n.a.	n.a.	n.a.
IC50[µM]	686.74	730.18	n.a.	n.a.	n.a.

n.a.: not applicable

Acceptance Criteria

Table7: Acceptance Criteria

Criterion	Range	Exp. 1	pass/fail	Exp. 2	pass/fail	Exp. 3	pass/fail
CV Solvent Control	< 20%	13.9	pass	15.8	pass	14.9	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	2.0	pass	2.0	pass
EC1.5 PC	±2 x SD of historical mean	16.67	pass	27.36	pass	20.08	pass
Induction PC at 64 µM	2 .00 < x < 8.00	3.27	pass	2.52	pass	3.15	pass

Exp.: Experiment

Historical Data

Table8: Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.5	3.5	174
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	174
EC1.5 PC	7 < x < 34 µM	18.9	5.9	174
Induction PC at 64 µM	2.00 < x < 8.00	3.8	1.4	174

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

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.

In the present study FMOC-HIS-AIB-OH TFA LS was dissolved in DMSO. Based on a molecular weight of 576.51 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 1.92 was determined at a test item concentration of31.25 µM. The corresponding cell viability was92.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.88) was found to be 15.63 µM. The corresponding cell viability was >70% (89.1%).The calculated EC_1.5was<1000 µM (9.89 µM).

In the first experiment, no dose response for luciferase activity induction was observed and the test item is therefore considered as inconclusive.

In the second experiment,nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated. Therefore, the test itemcan be considered asnon-sensitiser in the second experiment.

With two different outcomes the guideline[4]recommends to do a third experiment for classification.

In the third experiment,nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

No dose response for luciferase activity induction was observed for two independent runs as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13/02/2019 - 05/06/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (IN VITRO SKIN SENSITISATION: HUMAN CELL LINE ACTIVATION TEST (H-CLAT))

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

Batch: 160004
Preparation date: 30/11/2016
Expiration date: 09/02/2020
Storage conditions: Refrigerated (2-8 °C)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The test was carried out on the human cell line THP-1, in two independent runs. Cells were exposed to 8 different concentrations of the test chemical for 24 hours. CD54/CD86 over-expression, strictly related to the sensitizing potential of the test substance, was assessed by flow cytometry.

Vehicle / solvent control:

other: As solvent controls were employed: - NC: culture medium - NC+PS: culture medium with physiological solution (50 μL of physiological solution in 2.45 mL of culture medium) - NC+DMSO: culture medium with DMSO (10 μL of DMSO in 2.49 mL of culture medium)

Negative control:

other: lactic acid

Positive control:

other: 2,4-dinitrochlorobenzene (DNCB) and nickel sulfate hexahydrate (NiSO4 6H2O) were used as positive controls

Key result

Group:

test chemical

Run / experiment:

other: other: Run 1/test dose in well: 500 µg/ml

Parameter:

other: RFI CD54

Value:

113.99 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

other: other: Run 2/test dose in well: 500 µg/ml

Parameter:

other: RFI CD54 (%)

Value:

70.13 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

other: other: Run 1/test dose in well: 500 µg/ml

Parameter:

other: RFI CD86 (%)

Value:

87.98 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

other: other: Run 2/test dose in well: 500 µg/ml

Parameter:

other: RFI CD86 (%)

Value:

67.79 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

other: NEGATIVE (non-sensitizer) up to the concentration 500 µg/ml

RFI calculation

The RFI was used as an indicator of CD54 and CD86 expression and it was calculated applying the following formula:

RFI = (MFI of chemical treated cells - MFI of chemica l isotype cells) /(MFI of solvent treated control cells - MFI of solvent treated isotype control cells) X 100

Where MFI = geometric mean fluorescence intensity.

Notably, “chemical/solvent treated cells” corresponds to cells labelled with CD54 or CD86, ”chemical/solvent treated isotype cells” corresponds to IgG1 labelled cells.

For each concentration of the test substance the cell viability was recorded from the isotype control cells, i.e. those cells stained with FITC-labelled IgG1.

Collected data were analyzed with Kaluza Flow Cytometry analysis Software. The composites of the software named “CD54-CD86 Expression Assay test substance h-CLAT” and “CD54-CD86 Expression Assay controls h-CLAT” were used together with the set up “compensation h-CLAT SRA 508”. For the analysis the mean geometric fluorescence intensity (MFI) of viable cells and viability for each sample were used. Relative Fluorescence Intensity (RFI) values were calculated using the 607/GxP data sheet. When cell viability is less than 50 % the relative fluorescence intensity (RFI) was not used because of the diffuse labeling of cytoplasmatic structures that are generated due to cell membrane destruction.

The results of CD54/CD86 expression assay are reported in Table:

	Test dose in well (μg/ml)	RFI CD54 (%)		RFI CD86 (%)		Viability (%)
		Run 1	Run 2	Run 1	Run 2	Run 1	Run 2
Test substance C1	500.00	113.99	70.13	87.98	67.79	97.65	97.46
Test substance C2	416.67	88.81	91.31	116.22	86.73	97.72	97.33
Test substance C3	347.23	85.16	146.60	118.73	86.51	97.53	97.46
Test substance C4	289.36	82.34	83.68	59.94	88.02	97.75	97.69
Test substance C5	241.13	82.15	84.93	78.68	94.94	97.77	97.49
Test substance C6	200.94	88.37	100.05	79.29	99.56	97.78	97.54
Test substance C7	167.45	89.96	97.16	86.64	97.55	97.89	97.50
Test substance C8	139.54	81.15	93.51	77.39	85.81	97.76	97.71
DNCB	4.00	399.92	508.10	380.71	346.25	84.54	75.10
NC	/	/	/	/	/	97.92	97.94
NC+PS	/	81.17	86.89	95.69	95.30	97.93	97.86
NC+DMSO	/	61.38	85.18	91.34	96.19	98.04	97.69

 

EC200 and EC150 determination

For test substances to be considered sensitizers two effective concentrations (EC values) EC200 for CD54 and EC150 for CD86 were also calculated.

These values indicate the concentrations at which the test substance induces a RFI of 200 (CD54) or 150 (CD86).

EC200 and EC150 were calculated by linear interpolation using the 607/GxP data sheet, applying the following formula:

EC200 (for CD54) = Bdose + [(200 - BRFI) / (ARFI - BRFI) × (Adose - Bdose)]

And

EC150 (for CD86) = Bdose + [(150 - BRFI) / (ARFI - BRFI) × (Adose - Bdose)]

Where:

Adose is the lowest concentration in μg/ml with RFI > 200 % (CD54) or 150 % (CD86)

Bdose is the highest concentration in μg/ml with RFI < 200 % (CD54) or 150 % (CD86)

ARFI is the RFI at the lowest concentration with RFI > 200 % (CD54)or 150 % (CD86)

BRFI is the RFI at the highest concentration with RFI < 200 % (CD54) or 150 % (CD86)

In the CD54/CD86 expression assay, RFI of CD54 is < 200 % and RFI CD86 is < 150 % at any tested dose, therefore it was not possible to calculate the EC200 and EC150 values.

CONCLUSIONS

When the RFI of CD54 is ≥ 200 % at any tested dose contemporary showing cell viability > 50 %, in the two independent runs, AND/OR if the RFI of CD86 is ≥ 150 % at any tested dose contemporary showing cell viability > 50 %, in the two independent runs, the test chemical is considered POSITIVE (sensitizer), otherwise it is considered NEGATIVE (non-sensitizer). If the two independent runs are not concordant for at least one of the markers (CD54 or CD86), it is necessary to perform and additional run and conclusions are based on the majority result of the three individual runs (i.e., 2 out of 3).

In the present study, the test chemical “FMOC-HIS-AIB-OH TFA LS; N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-histidyl-2-methylalanine and trifluoroacetic acid (1:1)”, CAS 1446013-08-6, batch n° 160004 assayed is to be considered NEGATIVE (non-sensitizer) up to the concentration 500 μg/ml.

Since 500 μg/ml in DMSO (highest soluble concentration) was used as the maximal test concentration in the CD54/CD86 expression assay, negative result is acceptable even if the cell viability was above 90 % at the highest concentration.

A third run was not necessary, since the first 2 runs produced concordant results.

Interpretation of results:

GHS criteria not met

Conclusions:

In the present study, the test chemical “FMOC-HIS-AIB-OH TFA LS; N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-histidyl-2-methylalanine and trifluoroacetic acid (1:1)”, CAS 1446013-08-6, batch n° 160004 assayed is to be considered NEGATIVE (non-sensitizer) up to the concentration 500 μg/ml.
Since 500 μg/ml in DMSO (highest soluble concentration) was used as the maximal test concentration in the CD54/CD86 expression assay, negative result is acceptable even if the cell viability was above 90 % at the highest concentration.

Executive summary:

The test substance “FMOC-HIS-AIB-OH TFA LS; N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-histidyl-2-methylalanine and trifluoroacetic acid (1:1)”, CAS 1446013-08-6, batch n° 160004 was subjected to h-CLAT assay conducted according to SOPa 120 rev.5 and based on OECD 442E:2018.

Nature of the study: short-term study.

The test was carried out on the human cell line THP-1, in two independent runs. Cells were exposed to 8 different concentrations of the test chemical for 24 hours. CD54/CD86 over-expression, strictly related to the sensitizing potential of the test substance, was assessed by flow cytometry.

Under the test conditions above described, the test substance “FMOC-HIS-AIB-OH TFA LS; N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-histidyl-2-methylalanine and trifluoroacetic acid (1:1)”, CAS 1446013-08-6, batch n° 160004 resulted to be NEGATIVE (NON-SENSITIZER) up to the maximal tested concentration of 500μg/mL.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)