3-hydroxypropyl octanoate

Endpoint summary

Description of key information

There was no reactivity and no peptide depletion in the presence of the test item hence it is classed as “no to minimal reactivity” and is predicted as a non-skin sensitizer by DPRA.

In the h-CLAT study the test item is considered to have a skin sensitization potential.

In the KeratinoSens assay study the test item is considered to have no skin sensitization potential.

Based on the results of the in vitro/in chemico studies the test item is considered to have no skin sensitization potential.

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:

2017-04-24 to 2017-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.

Main test:
The test item was tested in two independent runs.
For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75 (first XTT CV75: 384.6μg/mL; second XTT CV75: 256.7μg/mL; mean CV75: 320.65 μg/mL). Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
During flow cytometry acquisition dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).

Acceptance Criteria
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control is adjusted to 100 % and the cell viability of the DMSO control should be more than 90 % in comparison to the medium control.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105 %.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the cell viability should be > 50%.
• For the test chemical, the cell viability should be more than 50 % in at least four tested
concentrations in each run.

Negative results are acceptable only for test items exhibiting a cell viability of < 90 % at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 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. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90 %.

Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs:
− The RFI of CD86 is ≥ 150 % at any tested concentration (with cell viability ≥ 50 %);
− The RFI of CD54 is ≥ 200 % at any tested concentration (with cell viability ≥ 50 %).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE.

Key result

Run / experiment:

other: other: 2; 321 μg/mL

Parameter:

other: RFI of CD54

Value:

206.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: other: 2; 223 μg/mL

Parameter:

other: RFI of CD86

Value:

158

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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

The CV75 value of the first XTT test: 384.6 μg/mL

The CV75 value of the second XTT test: 256.7 μg/mL

The mean CV75 value of both XTT tests: 320.65 μg/mL

Table 1: Results of h-CLAT runs

	Concentration (μg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
1st h-CLAT run
Medium Control	-	100	100	100
DMSO Control	-	100	100	100
Positive Control (DNCB)	2	219#	711.7#	80.6
	3	358.3#	734.4#	73
Test Item	107	113.8	124.4	104
	129	114.4	101.9	105.8
	155	109	109.6	102.4
	186	129.3	131.4	99.3
	223	150.9	151.3#	97.6
	267	160.5	169.9#	97.2
	321 E	224#	549.4#	37.5
	385 E	345.5#	3460.3#	3.8
2nd h-CLAT run
Medium Control	-	100	100	100
DMSO Control	-	100	100	100
Positive Control (DNCB)	2	229.6#	396.4#	81.7
	3	219.9#	385.1#	77.9
Test Item	107	115.6	101.3	105.4
	129	112.8	112.1	105.8
	155	125.6	114.6	106.4
	186	146.1	136.9	105.9
	223	173.3	158#	108.3
	267	188.9	179#	104
	321	206.7#	343.9#	70.9
	385 E	272.2#	1317.2#	13.9

E=cell viability below 50 %, are excluded from the evaluation

#=RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %)

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In the h-CLAT study the test item is considered to have a skin sensitization potential.

Executive summary:

An in vitro Human Cell Line Activation Test (h-CLAT) according to OECD 442E was performed to assess the skin sensitizating potential of the test item dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 625 μg/mL in the first XTT test and of 312.5 μg/mL in the second XTT test up to the highest tested concentration (5000 μg/mL). The mean CV75 value of both XTT tests was calculated as 320.65 μg/mL. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 107, 129, 155, 186, 223, 267, 321 and 385 μg/mL

The test item with a calculated log Pow of 2.84 was tested in 2 independent runs. The cell viability of the two highest tested test item concentrations of the first run and the highest concentration of the second run were below 50 % and therefore excluded from the evaluation. The RFI of CD86 was equal or greater than 150 % in at least one concentration of both independent run data. In addition, the RFI of CD54 was greater than 200 % in one tested concentration of the second run. Both runs were POSITIVE relating to the prediction model used in the h-CLAT test method, therefore the test item is considered to have a skin sensitization potential. In the DMSO solvent control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) and the cell viability was >50 %. In conclusion, the test item activated THP-1 cells under the test conditions of this study. Therefore the test item is considered to be positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-07-20 to 2018-01-16

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:

2015-02-04

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

- 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).

- Controls
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the 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.
Cinnamic aldehyde (CA) was used as positive control. CA was dissolved in DMSO with a final concentration range of 4 µM - 64 µM.

- 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 6 experiment 1; P 8 experiment 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1 °C and 5 % CO2. For test material exposure, cells were cultured in medium.

- Luciferase Assay System
The luciferase activity was determined using Luciferase Assay System 10-Pack kit (Promega, Cat. No.: El501, Lot No.: 0000246522) and Luciferase Cell Culture Lysis 5x Reagent kit (Promega, Cat. No.: El531, Lot No.: 0000265318).

- 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.

- Experimental Procedure:
A cell suspension of 8 x 10^4cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 * 10^4 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 crosscontamination 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 (Gibco Life Science; Lot No.: 1877596). 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 1.000 ms before assessing the luciferase activity for 2.000 ms. 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) or over the weekend (experiment 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

- Prediction Model
The test item is considered positive in accordance with UN GHS “Category 1” 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. 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 which is consisting of 6 wells.

Key result

Run / experiment:

other: other: 1+2

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

EC1.5: 12.44 µM in experiment 1; 16.09 µM in experiment 2

Remarks on result:

no indication of skin sensitisation

Remarks:

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

Table 1: Induction of Luciferase Activity - Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1	1	1	0
Positive Control	4	1.29	1.17	1.23	0.09
	8	1.3	1.26	1.28	0.03
	16	1.66	1.5	1.58	0.12	*
	32	2.11	2.09	21	0.01	*
	64	4.18	3.95	4.06	0.16	*
Test Item	0.98	0.89	0.87	0.88	0.01
	1.95	0.9	0.98	0.94	0.06
	3.91	0.88	0.88	0.88	0
	7.81	0.93	0.87	0.9	0.05
	15.63	0.89	0.89	0.89	0
	31.25	0.84	0.9	0.87	0.04
	62.5	0.83	0.87	0.85	0.03
	125	0.77	0.92	0.86	0.13
	250	0.71	1.21	0.96	0.36
	500	0.79	1.02	0.9	0.16
	1000	0	0	0	0
	2000	0	0	0	0

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

Table 2: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5	n/a	n/a	-	-
Imax	0.93	1.21	1.07	0.2
IC30	398.62	152.75	275.67	173.88
IC50	492.32	311.09	401.71	128.15

n/a=not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

In the KeratinoSens assay study the test item is considered to have no skin sensitization potential.

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 the test item was dissolved in DMSO. Based on a molecular weight of 202 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 and a stable suspension was formed. 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, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. 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.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser.

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

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-04-06 to 2017-04-13

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

- Synthetic peptide containing Cysteine
Alternative name: Ac-RFAACAA-OH
Batch and Supplier: 1556171, AnaSpec
Purity: 95 % (by HPLC)
Molecular Weight: 751.5 g/mol

- Synthetic peptide containing Lysine
Alternative name: Ac-RFAAKAA-OH
Batch and Supplier: 1556172 , AnaSpec
Purity: 94 % (by HPLC)
Molecular Weight: 776 g/mol

- Positive control: Cinnamic Aldehyde

The solubility of the test item in acetonitrile was assessed at a concentration of 100 mM.

- Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

- Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

- Preparation of Stability Controls and Precision Controls
Stability controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile.

- Preparation of Positive Control Stock Solution and Test Item Stock Solution
100 mM stock solutions in acetonitrile of the positive control chemical (Cinnamic Aldehyde) and the test item were prepared.

- Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of the test item and the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either test item or positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

- Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of the test item and the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either test item or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

- Incubation
The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25 °C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

- Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of test item and the associated positive controls was quantified by HPLC using UV detection.
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile Phase (MP) A: 0.1 % TFA in Water
Mobile Phase (MP) B: 0.085 % TFA in ACN
Gradient:
Time (minutes) MP A (%) MP B (%)
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
Flow rate: 0.35 mL/minute
Stroke volume: 25 µL
Detector wavelength: UV, 220 nm
Injection volume: 2 μL (slow draw rate)
Run time: 30 min
Approximate retention time (Cysteine): 11 min
Approximate retention time (Lysine): 7 min

Positive control results:

The mean peptide depletion value for the positive control was 72.3 % for Cyteine and 58.4 % for Lysine. The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 60.8-100 % (SD < 14.9 %) and a mean lysine peptide depletion value of 40.2-69.0 % (SD < 11.6 %).

Key result

Run / experiment:

mean

Parameter:

other: Mean peptide depletion (Lysine)

Value:

-0.044

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: Mean peptide depletion (Cysteine)

Value:

-0.213

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Table 1: Analytical acceptance criteria

	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r^2>0.99	60.8-100 (SD <14.9 %)	0.45-0.55 mM (CV <15 %)	SD <14.9 %
	Lysine	r^2>0.99	40.2-69.0 (SD <11.6 %)	0.45-0.55 mM (CV <15 %)	SD<11.6 %
Achieved results	Cysteine	r^2>0.999	72.3 (SD, 0.01 %, n=3)	B: 0.505 mM (CV 1.08 %, n=6)	SD 0.20 % (n=3)
	Lysine	r^2>0.999	58.4 (SD, 0.41 %, n=3)	B: 0.506 mM (CV 0.34 %, n=6)	SD 0.30 % (n=3)

CV=Coefficient of Variation

SD=Standard deviation

 

Table 2: Depletion of peptide by test item

	Mean peak area of reference control (μV.sec)	Mean peak area of peptide with test item (μV.sec)	Mean peptide depletion by test item (%)
Cysteine	Control B: 881820 (n=6)	883700 (n=3)	-0.213
Lysine	Control B: 756720 (n=6)	757050 (n=3)	-0.044

Interpretation of results:

GHS criteria not met

Conclusions:

There was no reactivity and no peptide depletion in the presence of the test item hence it is classed as “no to minimal reactivity” and is predicted as a non-skin sensitizer by DPRA.

Executive summary:

The purpose of the study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item.

Solutions of the test item were successfully analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. There was no reactivity (no depletion) in the presence of either peptide which places the test item in the reactivity class of “no or minimal reactivity” and therefore it is predicted by DPRA to be a non-skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

To assess the skin sensitisation potenial of the test substance a weight of evidence approach was conducted using in vitro and in chemico methods.

 

DPRA

The purpose of the study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item.

Solutions of the test item were successfully analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. There was no reactivity (no depletion) in the presence of either peptide which places the test item in the reactivity class of “no or minimal reactivity” and therefore it is predicted by DPRA to be a non-skin sensitizer.

 

h-CLAT

An in vitro Human Cell Line Activation Test (h-CLAT) according to OECD 442E was performed to assess the skin sensitizating potential of the test item dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 625 μg/mL in the first XTT test and of 312.5 μg/mL in the second XTT test up to the highest tested concentration (5000 μg/mL). The mean CV75 value of both XTT tests was calculated as 320.65 μg/mL. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 107, 129, 155, 186, 223, 267, 321 and 385 μg/mL

The test item with a calculated log Pow of 2.84 was tested in 2 independent runs. The cell viability of the two highest tested test item concentrations of the first run and the highest concentration of the second run were below 50 % and therefore excluded from the evaluation. The RFI of CD86 was equal or greater than 150 % in at least one concentration of both independent run data. In addition, the RFI of CD54 was greater than 200 % in one tested concentration of the second run. Both runs were POSITIVE relating to the prediction model used in the h-CLAT test method, therefore the test item is considered to have a skin sensitization potential. In the DMSO solvent control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) and the cell viability was >50 %. In conclusion, the test item activated THP-1 cells under the test conditions of this study. Therefore the test item is considered to be positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

 

KeratinoSens

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 the test item was dissolved in DMSO. Based on a molecular weight of 202 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 and a stable suspension was formed. 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, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. 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.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser.

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

 

Conclusion

Based on the results of an in chemico/in vitro test strategy the test item shows no or minimal peptide reactivity (DPRA, OECD TG 442C) but it does activate dentritic cells (h-CLAT, OECD TG 442E). Therefore, a third study was conducted and this KeratinoSens test (OECD TG 422D) showed no activation of keratinocytes. Overall those test results lead to the prediction that the substance is no 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

Based on available data on skin sensitisation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.