Carnosine

Endpoint summary

Description of key information

Skin sensitisation (OECD 442 C): negative

Skin sensitisation (OECD 442 D): negative

Skin sensitization (OECD 442 E): negative

WoE conclusion from in vitro skin sensitisation battery: not skin sensitising

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:

22 May 2017 - 27 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: AnaSpec
- Synthetic cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/mol
Purity: 95%
Expiry date: 5 years
- Synthetic lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/mol
Purity: 94%
Expiry date: 5 years

VEHICLE CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Vehicle (positive control): acetonitrile
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile.
- Vehicle (test item): water
The solubility of the test substance in a series of solvents was assessed at a concentration of 100 mM. A stock solution of the test substance was prepared at 100 mM in water.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Batch number: MKBR2427V
- Purity: > 95%
- Expiry date: Feb 2019

STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance/positive control); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance/positive control)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h prior to initiation of the analysis run

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with guard column Phenomenex AJO4286
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 20, 21, 23, 23.5, 30
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0.0167, 0.0334, 0.0667, 0.133, 0.267 and 0.534 mM
- Column temperature: 30 °C
- Sample temperature: 25 °C

Parameter:

other: % depletion of cystein-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

0.075

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Parameter:

other: % depletion of lysine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

1

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Parameter:

other: % overall mean depletion

Value:

0.536

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution peaks were observed in either the Cysteine or Lysine assay. The solubility of the test substance in water at a nominal concentration of 100 mM was confirmed.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide were met.

Table 5: Depletion of cysteine-containing peptide

Sample	Peak area (µV.sec)	Peptide concentration^*(µg/mL)	Peptide Depletion^**(%)	Mean Depletion (%)	SD (%)
Positive control	256166	112.78	70.1	70.0^***	0.30
	256241	112.81	70.1
	260636	114.75	69.6
Test substance^***	858192	378.14	-0.481	0.0750	0.61
	847846	373.58	0.730
	854295	376.43	-0.0250

SD    Standard Deviation

^*             Samples prepared at a concentration of 376 µg/mL (0.5 mM)

^**           Calculated against a mean Reference Control area of 857880 µV.sec(n=6)

^***          Individual and mean concentration values are outside of the range set in the historic data (Annex 1). This is considered not to have impacted on the data reported as the mean depletion value is within acceptance criteria limits.

^****        Calculated against a mean Control C peak area of 854080 µV.sec(n=3)

Table 6: Depletion of lysine-containing peptide

Sample	Peak area (µV.sec)	Peptide concentration^*(µg/mL)	Peptide Depletion^**(%)	Mean Depletion (%)	SD (%)
Positive control	305750	158.56	60.9	59.8	0.99
	316448	163.91	59.6
	320860	166.12	59.0
Test substance^***	766486	389.04	0.113	1.00	1.07
	762062	386.82	0.689
	750596	381.09	2.18

SD   Standard Deviation

^*             Samples prepared at a concentration of 388 µg/mL (0.5 mM)

^**      Calculated against a mean Reference Control area of 782340 µV.sec(n=6)

^***     Calculated against a mean Control C peak area of 767350 µV.sec(n=3)

 

Interpretation of results:

other: DPRA prediction: negative (no or minimal peptide reactivity) according to OECD 442C

Conclusions:

Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity.

Executive summary:

Protein reactivity of the test substance was determined in a Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C and in compliance with GLP (2017). The overall mean percentage of cystein and lysine depletion was 0.536% and therefore the test substance showed no or minimal peptide reactivity (mean % depletion ≤ 6.38%).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 May - 19 Oct 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:

adopted 4 Feb 2015

Deviations:

yes

Remarks:

No third repetition, although the first two repetitions were not concordant (first experiment: negative, second experiment: inconclusive)

Qualifier:

according to guideline

Guideline:

other: KeratinoSensTM, FURL ECVAM DB-ALM

Version / remarks:

Protocol No. 155
1 Jul 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of study:

activation of keratinocytes

Details on the study design:

TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance 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 sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 3 (Experiment 1), 5 (Experiment 2)

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-pyruvate, 10% fetal bovine calf serum and 1% geneticin (final concentration 500 µg/mL)
Assay medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-pyruvate and 10% fetal bovine calf serum
Test substance exposure medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-pyruvate and 1% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

TEST CONCENTRATIONS
0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000 and 2000 µM

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1% (v/v)
Positive control
- Substance: cinnamic aldehyde
- Final concentration: 4 - 64 µM in DMSO

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 1 h at 37 °C ± 1 °C and 5% CO2

NUMBER OF REPLICATIONS: One well of each test substance or positive control concentration and six wells of vehicle control were measured in triplicates in two independent experiments

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 600 nm

DETERMINATION OF LUMINESCENCE
- Cell lysis reagent: Luciferase Cell Culture Lysis 5x Reagent Kit (Promega, Cat. No. E1531, Lot No. 0000087405)
- Luciferase reagent: Luciferase Assay Kit (Promega, Cat. no. E1501, Lot No. 00002375336)
- Device: plate reader

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: maximum luciferase activity

Value:

1.27

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 2000 µM; cell viability: 87.9%

Run / experiment:

other: Experiment 2

Parameter:

other: maximum luciferase activity

Value:

1.98

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: test item concentration: 62.50 µM; cell viability: 64.3%

Other effects / acceptance of results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment 1: No evidence of cytotoxicity (cell viability < 70%) was observed at all test substance concentrations.
In experiment 2: cell viability was < 70% at test substance concentrations of 62.5, 125, 250, 500, 1000 and 2000 µM.

In experiment 2 induction of the luciferase activity above 1.5 could already be observed starting at a concentration of 0.98 µM, oscillating around the threshold of 1.5 in the following concentration steps. Since the induction follows no dose response relationship, this induction was considered to be not biologically relevant. No EC1.5 was calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: The average coefficient of variation of the luminescence reading for the vehicle control was < 20% (7.0% and 17.8.% in Experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 ( 2.36 and 3.26 in Experiment 1 and 2, respectively) and the calculated EC1.5 value was between 7 and 34 µM (23.95 and 20.35 µM in Experiment 1 and 2, respectively).

Table 2: Results of the cytoxicity measurement

	Concentration [µM]	Cell Viability[%]
		Experiment 1	Experiment 2	Mean ± SD
Vehicle Control	-	100	100	100 ±0.0
Positive Control	4.00	101.8	92.5	97.2 ±6.5
	8.00	105.5	99.0	102.3 ±4.6
	16.00	113.2	93.9	103.5 ±13.6
	32.00	116.8	91.4	104.1 ±17.9
	64.00	125.4	85.0	105.2 ±28.5
Test Substance	0.98	95.9	87.9	91.9 ±5.6
	1.95	96.7	98.3	97.5 ±1.1
	3.91	91.6	81.2	86.4 ±7.4
	7.81	91.6	85.7	88.6 ±4.2
	15.63	97.1	82.8	89.9 ±10.2
	31.25	90.4	83.4	86.9 ±5.0
	62.50	84.5	64.3	74.4 ±14.3
	125.00	82.9	63.9	73.4 ±13.4
	250.00	83.1	57.4	70.2 ±18.2
	500.00	87.5	57.1	72.3 ±21.5
	1000.00	81.9	46.9	64.4 ±24.7
	2000.00	87.9	46.1	67.0 ±29.5

Table 3. Induction of luciferase activity

Overall lnduction	Concentration (µM)	Fold lnduction
		Experiment 1	Experiment 2	Mean ± SD
Vehicle Control	-	1.00	1.00	1.00 ± 0.00
Positive Control	4.00	1.10	1.10	1.10 ± 0.00
	8.0	1.15	1.34	1.24 ± 0.13
	16.00	1.39	1.35	1.37 ± 0.03
	32.00	1.61	1.91	1.76 ± 0.21*
	64.00	2.36	3.26	2.81 ± 0.64
Test Substance	0.98	1.18	1.67	1.43 ± 0.35
	1.95	1.11	1.73	1.42 ± 0.44
	3.91	1.19	1.52	1.35 ± 0.23
	7.81	1.05	1.58	1.31 ± 0.38
	15.63	1.19	1.64	1.42 ± 0.32
	31.25	1.14	1.34	1.24 ± 0.14
	62.50	1.17	1.98	1.57 ± 0.58
	125.00	1.25	1.36	1.31 ± 0.07
	250.00	1.19	1.71	1.45 ± 0.36
	500.00	1.17	1.37	1.27 ± 0.14
	1000.00	1.22	1.38	1.30 ± 0.12
	2000.00	1.27	1.28	1.27 ± 0.00

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

Interpretation of results:

other: activation of keratinocytes negative

Remarks:

according to OECD 442D

Conclusions:

Under the conditions of the ARE-Nrf2 Luciferase test the test substance did not induce luciferase activity in the transgenic KeratinoSens™ cell line.

Executive summary:

The activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test (LuSens) in the transgenic KeratinoSens™ cell line according to OECD Guideline 442D and in compliance with GLP (2017). In the first experiment, a max luciferase activity induction (Imax) of 1.27 was determined at a concentration of 2000 µM. The corresponding cell viability was 87.9%. In the second experiment, a max luciferase activity (Imax) induction of 1.98 was determined at a test item concentration of 62.50 µM. The corresponding cell viability was 64.3%. Induction of the luciferase activity above 1.5 could already be observed starting at a concentration of 0.98 µM, oscillating around the threshold of 1.5 in the following concentration steps. Since the induction followed no dose response relationship, this induction was considered to be not biologically relevant. No EC1.5 was calculated. The test substance did not induce luciferase activity in the transgenic KeratinoSensTM cell line in at least two independent experimental runs.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 April - 12 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

cytotoxicity measurement and estimation of the CV75 value was performed by XTT test instead of flow cytometry

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

activation of dendritic cells

Details on the study design:

TEST CELL LINE
- Strain: THP-1 cells (human monocytic leukemia cell line)
- Source: American Type Culture Collection (ATCC), # TIB-202
- Passage number: 17 (XTT test); 21 and 25 (h-CLAT)

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and 1% (v/v) L-glutamine
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5.0 ± 0.5

DOSE FINDING ASSAY BY CYTOTOXICITY MEASUREMENT (XTT TEST)
The doses investigated in the main experiment (h-CLAT) were determined with a XTT test. The XTT test is a method to investigate the cytotoxicity of a test substance. The test is based on the cleavage of the yellow tetrazolium salt, sodium 3'-(1-phenylaminocarbonyl)-(3,4 - tetrazolium)-bis-(4–methoxy-6-nitro)-benzenesulfonic acid hydrate (XTT), to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable concentration showing 75% cell viability (CV75). The highest soluble test item concentration of 5000 µg/mL was used to prepare seven 1:2 serial dilutions in culture medium.

CONTROLS
Negative control
- Substance: culture medium
Vehicle control (for the positive control)
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 0.2%

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 0.5 h

TEST CONCENTRATIONS
- Justification for top dose: The test substance was dissolved in culture medium. The highest soluble test item concentration of 5000 µg/mL was used to prepare seven 1:2 serial dilutions in culture medium.

39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL

NUMBER OF REPLICATIONS: septuplicate (7 wells) per concentration in two independent experiments

MEASUREMENT
- Device: microplate reader (Versamax Molecular Devices)
- Wavelength: 450 nm
- Reference wavelength: 690 nm

EVALUATION CRITERIA
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance as compared to the solvent control is calculated using the formula:
Relative absorbance [%] = 100 x [(mean absorbance test substance) - (absorbance blank)] / [(mean absorbance vehicle control) - (absorbance blank)]

To calculate the concentration of test substance needed to reduce the relative absorbance to 75% of the vehicle control (CV75) the following formula is used:
CV75 = Conc. > 75 - ([(Conc. > 75) - (Conc. < 75)] x [(% > 75) - 75] / [(% > 75) - (% < 75)])
a) Conc. > 75: max. measured concentration with the % of vehicle control > 75%
b) Conc. < 75: min. measured concentration with the % of vehicle control < 75%
c) % > 75: relative absorbance at a) in %
d) % < 75: relative absorbance at b) in %

ACCEPTANCE CRITERIA
The XTT test is considered to be acceptable if it meets the following criteria:
- mean absorbance of the negative control is ≥ 0.5
- mean viability of the vehicle control is ≥ 90% in comparison to the medium control

h-CLAT (main study)
The h-CLAT method is an in vitro method which quantifies changes of cell surface marker expression (CD86 and CD54) on a human cell line, THP-1 cells, following 24 h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies. The relative fluorescence intensity of surface markers compared to vehicle control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

CONTROLS
Negative control
- Substance: culture medium
Vehicle control (for positive control)
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2%
Positive control
- Substance: 2,4-dinitrochlorobenzene (DNCB) in DMSO
- Concentration: 2 and 3 µg/mL

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 0.5 h

TEST CONCENTRATIONS
- Justification for top dose: Since a CV75 value could not be determined in the XTT test, the highest test item concentration of 5000 μg/mL recommended by the OECD 442E guideline was used for the main study (h-CLAT). Seven dilutions were prepared by serial 1:1.2 dilution.
1395, 1674, 2009, 2411, 2894, 3472, 4167 and 5000 µg/mL

NUMBER OF REPLICATIONS: triplicates for the different stainings in two independent experiments

STAINING
- Antibodies: FITC anti-CD54 (Clone: Fun-1); FITC anti-CD86 (Clone: 6.5B5); FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C
- Duration: 30 ± 5 min
- Cell viability staining: 5 µL 7AAD solution

MEASUREMENT
- Device: flow cytometer FACSCalibur (Becton Dickinson)

EVALUATION CRITERIA
The relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration:
RFI (%) = [(MFI of test substance treated cells) - (MFI of test substance treated isotype control cells) / (MFI of vehicle control cells) - (MFI of vehicle isotype control cells)] x 100
MFI: geometric mean fluorescence intensity (GeoMean)

The cell viability is calculated as follows for each concentration:
Cell viability (%) = (mean cytotox of vehicle control cells) / (mean cytotox of test substance treated cells) x 100
Where the mean cytotox is the mean of GeoMean (7-AAD) isotype control, GeoMean (7-AAD) CD54 and GeoMean (7-AAD) CD86.

The test substance is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%) in both independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer. In case of different results for at least one of the markers, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test substance is considered to be a sensitizer. Also, If the third run is positive for only one of the markers the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer.

ACCEPTANCE CRITERIA
The study is considered as valid, if the following criteria are met:
- Cell viability of negative control is adjusted to 100% and the cell viability of the vehicle control should be more than 90% in comparison to the negative control.
- In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For negative and vehicle controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- In the positive control, RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
- For the test substance the cell viability sholud be more than 50% in at least four tested concentrations in each run.
- Negative results are only acceptable for the test substance exhibiting a cell viability < 90% at the highest concentration tested (i.e. 1.2 x CV75).
- If the cell viability at the 1.2 × CV75 value is ≥ 90% the negative result should be discarded. In such a case it is recommended to try to refine the dose selection by repeating the CV75 determination.
- If 5000 μg/mL in saline (or medium or other vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, instead of CV75-based dose, a negative result is acceptable even if the cell viability > 90%.

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: relative fluorescence intensity of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 24 h incubation

Parameter:

other: relative fluorescence intensity of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
XTT test
Cytotoxic effects were only observed in one out of two experiments, following incubation with the test substance up to the highest soluble concentration (5000 µg/mL). The viability of the cells spaced within a range of 69.18 to 98.25% in the first experiment and within a range of 81.79 to 106.83% in the second experiment (threshold of cytotoxicity: < 75%). The CV75 value of the first XTT test: 4499.5 µg/mL. Due to the lack of cytotoxicity in the second XTT test, a mean CV75 value could not be calculated.
h-CLAT
The cell viability was not negatively affected by the test substance (viability > 93%) in the 2 independent experiments.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The mean viability of the vehicle control in comparison to the medium control was 99.9% and 98.22% (Cell viability of the DMSO control should be > 90% in comparison to the medium control).
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 exceeded the positive criteria (CD86 >150% and CD54 >200%) and the cell viability was >50%.
- Acceptance criteria met for variability between replicate measurements: The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in both independent run data.
- For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype was more than 105%

Table 1: Results of the first XTT test

Test Group	Concentration [µg/mL]	Mean Absorbance* ± SD	Blank	Mean Absorbance - Blank	Absorbance in % of vehicle control**
Medium control	-	0.721 ± 0.051	0.195	0.526	100.01
Vehicle control	-	0.723 ± 0.060	0.197	0.526	100.00
Test substance	39.1	0.703 ± 0.062	0.200	0.503	95.71
	78.1	0.677 ± 0.077	0.204	0.473	89.98
	156.3	0.697 ±0.036	0.206	0.491	93.42
	312.5	0.681 ± 0.044	0.212	0.469	89.23
	625	0.738 ± 0.025	0.223	0.515	98.00
	1250	0.747 ± 0.022	0.235	0.511	97.25
	2500	0.803 ± 0.034	0.286	0.517	98.25
	5000	0.751 ± 0.020	0.387	0.364	69.18

* mean absorbance (absolute) of 7 wells

** relative absorbance (rounded values)

Table 2: Results of the second XTT test

Test Group	Concentration [µg/mL]	Mean Absorbance* ± SD	Blank	Mean Absorbance - Blank	Absorbance in % of vehicle control**
Medium control	-	0.588±0.032	0.200	0.388	101.81
Vehicle control	-	0.579±0.034	0.198	0.381	100.00
Test substance	39.1	0.561±0.039	0.198	0.363	95.20
	78.1	0.588±0.017	0.205	0.383	100.50
	156.3	0.584±0.025	0.208	0.376	98.79
	312.5	0.594±0.033	0.205	0.389	102.15
	625	0.607±0.039	0.221	0.386	101.38
	1250	0.640±0.041	0.233	0.407	106.83
	2500	0.641±0.034	0.281	0.361	94.63
	5000	0.690±0.027	0.378	0.312	81.79

* mean absorbance (absolute) of 7 wells

** relative absorbance (rounded values)

Table 3: Results of the first h-CLAT run

	Concentration [µg/mL]	Antibody / ISO	MFI GeoMean (FITC)	MFI - ISO	RFI (%)	Cyto (Geo) GeoMean (7-AAD)	Mean Cyto	Viability (%)
Medium control	-	ISO	1.80			2.92	2.5	100.0
		CD54	3.47	1.67	100.0	2.12
		CD86	3.36	1.56	100.0	2.31
Vehicle control	-	ISO	1.64			2.95	2.5	100.0
		CD54	3.27	1.63	100.0	2.13
		CD86	3.18	1.54	100.0	2.44
Positive control	2	ISO	2.09			4.30	3.1	80.6
		CD54	5.66	3.57	219.0*	2.98
		CD86	13.05	10.96	711.7*	2.05
	3	ISO	2.12			4.95	3.4	73.0
		CD54	7.96	5.84	358.3*	3.03
		CD86	13.43	11.31	734.4*	2.32
Test substance	1395	ISO	1.77			3.01	2.6	94.5
		CD54	3.12	1.35	80.8	2.39
		CD86	3.36	1.59	101.9	2.38
	1674	ISO	1.74			2.94	2.6	95.6
		CD54	2.97	1.23	73.7	2.44
		CD86	3.26	1.52	97.4	2.31
	2009	ISO	1.70			2.99	2.6	95.2
		CD54	2.91	1.21	72.5	2.41
		CD86	3.20	1.50	96.2	2.32
	2411	ISO	1.65			3.00	2.6	93.0
		CD54	2.90	1.25	74.9	2.47
		CD86	2.86	1.21	77.6	2.43
	2894	ISO	1.67			2.95	2.6	95.0
		CD54	2.94	1.27	76.0	2.39
		CD86	2.94	1.27	81.4	2.40
	3472	ISO	1.67			2.98	2.6	94.6
		CD54	2.88	1.21	72.5	2.37
		CD86	2.93	1.26	80.8	2.42
	4167	ISO	1.66			2.95	2.6	93.9
		CD54	3.02	1.36	81.4	2.43
		CD86	3.06	1.40	89.7	2.45
	5000	ISO	1.63			2.96	2.6	93.9
		CD54	3.00	1.37	82.0	2.43
		CD86	2.94	1.31	84.0	2.44

* RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥ 150% and CD54 ≥ 200%).

 

Table 4: Results of the second h-CLAT run

	Concentration [µg/mL]	Antibody / ISO	MFI GeoMean (FITC)	MFI- ISO	RFI (%)	Cyto(Geo) GeoMean (7-AAD)	Mean Cyto	Viability (%)
Medium control	-	ISO	1.54			2.56	2.3	100.0
		CD54	3.34	1.80	100.0	2.17
		CD86	3.11	1.57	100.0	2.07
Vehicle control	-	ISO	1.51			2.60	2.1	100.0
		CD54	3.47	1.96	100.0	1.82
		CD86	3.19	1.68	100.0	1.87
Positive control	2	ISO	1.76			3.52	2.6	81.4
		CD54	6.26	4.50	229.6*	2.34
		CD86	8.42	6.66	396.4*	1.87
	3	ISO	1.78			3.53	2.7	77.9
		CD54	6.09	4.31	219.9*	2.47
		CD86	8.25	6.47	385.1*	2.07
Test substance	1395	ISO	1.59			2.64	2.2	104.1
		CD54	3.26	1.67	92.8	1.93
		CD86	3.13	1.54	98.1	1.96
	1674	ISO	1.57			2.63	2.3	100.4
		CD54	2.96	1.39	77.2	2.07
		CD86	2.90	1.33	84.7	2.07
	2009	ISO	1.59			2.63	2.3	100.7
		CD54	3.03	1.44	80.0	2.09
		CD86	2.98	1.39	88.5	2.03
	2411	ISO	1.52			2.76	2.4	95.8
		CD54	3.03	1.51	83.9	2.14
		CD86	2.86	1.34	85.4	2.20
	2894	ISO	1.53			2.77	2.4	96.3
		CD54	3.02	1.49	82.8	2.15
		CD86	2.86	1.33	84.7	2.14
	3472	ISO	1.52			2.68	2.3	98.7
		CD54	3.01	1.49	82.8	2.10
		CD86	2.89	1.37	87.3	2.11
	4167	ISO	1.53			2.74	2.3	97.3
		CD54	3.07	1.54	85.6	2.10
		CD86	2.63	1.10	70.1	2.15
	5000	ISO	1.47			2.71	2.3	97.0
		CD54	2.82	1.35	75.0	2.16
		CD86	2.71	1.24	79.0	2.14

*: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥150% and CD54 ≥200%).

The test substance with a log Pow of -1.810 was tested in 2 independent runs. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in both independent run data. Both runs were NEGATIVE relating to the prediction model used in the h-CLAT test method, therefore the test item is considered to have no skin sensitization potential.

Interpretation of results:

other: activation of dendritic cells negative according to OECD 442E

Conclusions:

Under the conditions of the Human Cell Line Activation Test the test substance did not increase the expression of CD54 or CD86, cell surface markers associated with the process of activation of monocytes and dendritic cells. Based on this result, the h-CLAT prediction is considered negative for skin sensitisation.

Executive summary:

Dendritic cell response of the test substance was investigated by a Human Cell Line Activation Test (h-CLAT) according to OECD Guideline 442E and in compliance with GLP (2017). The test substance did not increase the expression of the cell suface marker CD54 and CD86 to greater than 200% and 150%, respectively in two independent experiments. Thus, the h-CLAT prediction for activation of dendritic cells is considered negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Protein reactivity of the test substance was determined in a Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C and in compliance with GLP (2017). The overall mean percentage of cystein and lysine depletion was 0.536% and therefore the test substance showed no or minimal peptide reactivity (mean % depletion ≤ 6.38%).

In a second study, the activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test (LuSens) in the transgenic KeratinoSens™ cell line according to OECD Guideline 442D and in compliance with GLP (2017). In the first experiment, a max luciferase activity induction (Imax) of 1.27 was determined at a concentration of 2000 µM. The corresponding cell viability was 87.9%. In the second experiment, a max luciferase activity (Imax) induction of 1.98 was determined at a test item concentration of 62.50 µM. The corresponding cell viability was 64.3%. Induction of the luciferase activity above 1.5 could already be observed starting at a concentration of 0.98 µM, oscillating around the threshold of 1.5 in the following concentration steps. Since the induction followed no dose response relationship, this induction was considered to be not biologically relevant. No EC1.5 was calculated. The test substance did not induce luciferase activity in the transgenic KeratinoSensTM cell line in at least two independent experimental runs.

In a third study, dendritic cell response of the test substance was investigated by a Human Cell Line Activation Test (h-CLAT) according to OECD Guideline 442E and in compliance with GLP (2017). The test substance did not increase the expression of the cell suface marker CD54 and CD86 to greater than 200% and 150%, respectively in two independent experiments. Thus, the h-CLAT prediction for activation of dendritic cells is considered negative.

Based on a weight of evidence approach considering three negative results in the in vitro test battery, the test substance is not considered to be a skin sensitiser.

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not indicate skin sensitisation potential for the test item and are conclusive but not sufficient for classification.

The data do not meet the criteria for classification according to Regulation (EC) 1272/2008.