Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate

Endpoint summary

Description of key information

The skin sensitisation potential of the test item sodium isostearoyl lactylate was assessed in two in vitro skin sensitisation assays conducted according to OECD 442D and OECD 442E as well as in one in chemico skin sensitisation assay conducted according to OECD 442C.

In the first study conducted according to OECD 442D with the test item dissolved in THF, the sensitisation potential of the test item was assessed based on the activation of keratinocytes using the in vitro KeratinoSens™ cell line. Based on this study, the test item is considered to be a skin sensitiser.

However, this result contrasts with the findings of the second study conducted according to OECD 442D with the test item dissolved in THF. In this study, the sensitisation potential of the test item was assessed based on the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). Based on the results of this study, the test item is not considered to be a skin sensitiser.

Lastly, in order to provide further evidence in a weight-of-evidence approach, the skin sensitisation potential of the test item sodium isostearoyl lactylate was assessed in chemico via the direct peptide reactivity assay, which showed minimal reactivity (or peptide depletion) of the test item to the cysteine or lysine peptide. Thus, this in chemico test demonstrated that the test item is not a skin sensitiser.

As an overall conclusion based on an assessment of the available data in a weight-of-evidence approach, sodium isostearoyl lactylate is not considered to be a skin sensitiser.

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:

2018-01-31 to 2018-07-24

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 04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitisers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore, the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Pationic ISL (Lot: 1731200025)
- Expiration date of the lot/batch: 12 months after opening

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: All test item solutions were freshly prepared immediately prior to use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in tetrahydrofuran (THF, purity ≥99%, CAS No.: 109-99-9, Sigma, Lot No.: STBH3251). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. A stable suspension was formed when diluted 1:100 in cell culture medium.
- Final dilution of a dissolved solid, stock liquid or gel: Based on the THF stock solution serial dilutions were made using the solvent (THF) to obtain 12 master concentrations of the test item (0.098 to 200 mM). The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the master solutions were further diluted 1:25 in cell culture medium.
These 1:25 diluted test item solutions were further diluted 1:4 when added to the cells so that the final concentrations of the test item range from 0.98 to 2000 µM. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and the respective solvent control.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

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 10 in experiment 1; P 9 in experiment 2) were used.

Cells were cultured in 75 cm^2 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 test item exposure medium.

LUCIFERASE ASSAY SYSTEM:
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacturer´s manual.

Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501, Lot No.: 0000277734) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilised)
- 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 equilibrated to room temperature prior to use.

Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000246522) consisted of 30 mL Luciferase Cell Culture Lysis 5x Reagent.
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBG3520).

DOSE GROUPS:
Solvent Controls: 1% (v/v) DMSO in test item exposure medium (for the positive control) 1% (v/v) THF in test item exposure medium (for the test item)
Positive Control: cinnamic aldehyde (4 μM; 8 μM; 16 μM; 32 μM; 64 μM)
Test Item: 12 serial concentrations (0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000, 2000 μM)

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The solvent controls were assessed using six replicates per 96-well plate in every independent run, respectively.

EXPERIMENTAL PROCEDURE:
The incubation was performed in 96-well plates. Cells were counted by Neubauer chamber and a cell suspension of 8 × 10^4 cells/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. Cells were mixed by swinging during pipetting into the 96-well plate to ensure homogeneous cell number distribution. 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 freshly prepared 25 times-diluted 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 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 (Tecan, Infinite 200Pro) for luminescence measurement. For each well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1000 ms before assessing the luciferase activity for 2000 ms. This procedure was repeated for each individual well of 96-well plate.

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. After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm using a plate reader (Tecan, Infinite 200Pro).

DATA ANALYSIS:
For each test item two independent runs 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.

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 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
The KeratinoSens™ prediction for skin sensitisation of the test item is considered positive if the following conditions are met in at least two independently prepared test runs:
- Imax is increased by >1.5-fold and is statistically significant (p<0.05) compared to the solvent control,
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5-fold,
- EC1.5 value is <1000 μM, and
- an apparent overall dose-response relationship for luciferase induction.
If in a given run, all of the three first conditions are met but a clear dose-response relationship 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.

Positive control results:

- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (7.40 in experiment 1; 3.90 in experiment 2).
- The calculated EC1.5 was between 7 and 34 µM (7.72 µM in experiment 1; 12.71 µM in experiment 2).

Key result

Run / experiment:

other: Experiment I at 15.63 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.64

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Experiment II at 15.63 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

2.06

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment I

Parameter:

other: EC1.5 [µM]

Value:

12.42

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment II

Parameter:

other: EC1.5 [µM]

Value:

7.18

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The acceptance criteria proposed by the OECD test guideline 442D were met in this test.

For individual results see Table 1 in box 'Any other information on results incl. tables'.

Table 1: Induction of Luciferase Activity - Overall Induction

	Concentration [µM]	Relative Fold Induction^1)
		Experiment 1	Experiment 2	Mean	SD
Positive Control	4.00	1.05	1.14	1.09	0.06
	8.00	1.53	1.29	1.41	0.17
	16.00	1.75	1.65	1.70	0.07
	32.00	3.81	2.13	2.97	1.18
	64.00	7.40	3.90	5.65	2.47
Test Item	0.98	1.04	1.24	1.14	0.14
	1.95	1.14	1.14	1.14	0.00
	3.91	1.16	1.18	1.17	0.02
	7.81	1.30	1.56	1.43	0.19
	15.63	1.64	2.06	1.85	0.30
	31.25	0.17	0.00	0.09	0.12
	62.50	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00

¹⁾ Percentage of fold induction is relative to the solvent control (i.e. set at 100%).

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study (performed in accordance with OECD 442D) under the given conditions the test item sodium isostearoyl lactylate induced the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, sodium isostearoyl lactylate can be considered as sensitiser in this test. However, based on the adverse outcome pathway (AOP) for skin sensitisation, a positive outcome in only one of the 3 key events is not sufficient for classification according to CLP.

Executive summary:

In a skin sensitisation study conducted according to OECD 442D, the sensitisation potential of the test item sodium isostearoyl lactylate was assessed on the basis of the activation of keratinocytes by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. Cells were incubated with the test item (12 concentrations tested, ranging from 0.98-2000 µM) 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 1.64 was determined at a test item concentration of 15.63 μM. The corresponding cell viability was >70% (115.4%). In the second experiment, a max luciferase activity (I_max) induction of 2.06 was determined at a test item concentration of 15.63 μM. The corresponding cell viability was >70% (107.8%). The lowest tested concentration with a significant luciferase induction >1.5-fold (1.56) was found to be 7.81 μM. The corresponding cell viability was >70% (107.0%). A dose-response relationship 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 sodium isostearoyl lactylate is therefore considered as sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-01-31 to 2018-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E: OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation)

Version / remarks:

Adopted 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The correlation of upregulation of immunologically relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event of the skin sensitisation process as described by the AOP for skin sensitisation. This method, which measures the markers of DC activation, using the DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Pationic ISL (Lot: 1731200025)
- Expiration date of the lot/batch: 12 months after opening

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: All test item solutions were freshly prepared immediately prior to use. The test item was soluble in tetrahydrofuran (THF) at a concentration of 500 mg/mL.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Vortex-mixing and warming to 37 °C were used to aid solubilisation. For the dose finding assay stock solutions were prepared by diluting the 500 mg/mL seven times with a constant dilution factor of 1:2. For the main experiments stock solutions were prepared by diluting the concentration of 72.62 mg/mL, which was determined to be the highest desired stock concentration based on the dose finding assay, seven times with a constant dilution factor of 1:2.

- Final dilution of a dissolved solid, stock liquid or gel: The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. No precipitation, turbidity or phase separation was observed when diluted 1:250 in cell culture medium.
The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (THF) was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

CELL LINE:
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for dendritic cells. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10^6 cells/mL.
Cells were cultured in 75 cm^2 culture flasks (Greiner) in cell culture medium consisting of Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin / 100 µg/mL streptomycin at 37 ± 1 °C and 5% CO2.

PRE-EXPERIMENTS:
Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 µg/mL and nickel sulphate (NiSO4) at a final concentration of 100 µg/mL served as positive controls while lactic acid (LA) at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both DNCB and NiSO4 produce a positive response and LA produces a negative response for the upregulation of CD86 and CD54.

Solvent Finding
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl at a final concentration of 100 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical was dissolved or stably dispersed in the chosen solvent and that it did not interfere with the test design.

EXPERIMENTAL PROCEDURE:
Dose Finding Assay
Using the 500 mg/mL stock solution of the test item, eight working stock solutions were prepared by 2-fold serial dilutions using the appropriate solvent (THF). These working stock solutions were further diluted 250-fold with cell culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then 500 µL of the cell suspension were seeded onto a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer.
200 µL of the cell suspension were transferred into a FACS tube and stained with a propidium iodide (PI) solution at a final concentration of 0.625 µg/mL.
The PI uptake of the cells (an indicator of cytotoxicity) was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 cells were acquired and cell viability was calculated for each test concentration.
The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate and set the concentration range of the test item for the main experiments.

CD54 and CD86 Expression
The test item was dissolved using THF as determined in the pre-experiment. Based on the concentration of the CV75 value determined in the dose finding assay 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then 500 µL of the cell suspension were seeded onto a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared on the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were incubated with 50 µL of FITC-labelled anti-CD86, FITC-labelled anti-CD54, or mouse FITC-labelled IgG1 (isotype) antibodies in the dark for 30 min at 4 °C. All antibodies were diluted in FACS buffer. After incubating and then washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).
The expression levels of CD86 and CD54 as well as cell viability (as determined by living cells with no PI uptake) were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was also calculated.

DATA ANALYSIS
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis such as calculations of the CV75, RFI, Effective Concentration 150 (EC150) and Effective Concentration 200 (EC200) values were performed using the software Microsoft Excel 2010 as appropriate. The mean values and standard deviations of the single replicates were determined using the respective Excel commands.

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

Positive control results:

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The thresholds of 150% for CD86 (333% experiment 1; 298% experiment 2) and 200% for CD54 (311% experiment 1; 269% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: % relative fluorescence intensity of CD86 at 100.86 µg/mL

Remarks:

Highest tested non-cytotoxic concentration

Value:

128

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: % relative fluorescence intensity of CD54 at 100.86 µg/mL

Remarks:

Highest tested non-cytotoxic concentration

Value:

106

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: % relative fluorescence intensity of CD86 at 100.86 µg/mL

Remarks:

Highest tested non-cytotoxic concentration

Value:

76

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: % relative fluorescence intensity of CD54 at 100.86 µg/mL

Remarks:

Highest tested non-cytotoxic concentration

Value:

96

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

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

For individual results see Table 1 in box "Any other information on results incl. tables".

Table 1: CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Relative Cell Viability [%]			Mean Fluorescence Intensity			Isotype IgG1 Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI) [%]		Ratio Isotype IgG1 to [%]
		CD86	CD54	Iso-type IgG1	CD86	CD54	Iso-type IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.7	96.3	95.8	2110	1080	627	1483	453	92	100	337	172
Solvent Control (0.2% THF)	-	95.1	95.8	95.5	2169	1033	631	1538	402	100	100	344	164
Solvent Control (0.2% DMSO)	-	95.9	96.2	96.4	2233	1070	617	1616	453	100	100	362	173
DNCB	4.00	85.4	86.0	84.6	6034	2063	655	5379	1408	333	311	921	315
Sodium isostearoyl lactylate	100.86	60.1	62.8	63.4	2649	1106	678	1971	428	128	106	391	163

Table 2 : CD54 and CD 86 Expression Experiment 2

Sample	Conc. [μg/mL]	Relative Cell Viability [%]			Mean Fluorescence Intensity			Isotype IgG1 Corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI) [%]		Ratio Isotype IgG1 to [%]
		CD86	CD54	Iso-type IgG1	CD86	CD54	Iso-type IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	93.5	93.7	91.9	4129	1482	864	3265	618	94	83	478	172
Solvent Control (0.2% THF)	-	92.1	92.2	92.1	3870	1438	854	3016	584	100	100	453	168
Solvent Control (0.2% DMSO)	-	92.7	93.2	92.8	4247	1537	788	3459	749	100	100	539	195
DNCB	4.00	75.1	74.4	74.7	10979	2696	682	10297	2014	298	269	1610	395
Sodium isostearoyl lactylate	100.86	76.6	77.6	77.6	3273	1536	976	2297	560	76	96	335	157

Interpretation of results:

GHS criteria not met

Conclusions:

In this study (performed in accordance with OECD 442E) under the given conditions the test item sodium isostearoyl lactylate did not trigger an upregulation of the expression of the cell surface markers CD86 and CD54 in two independent experiment runs. Therefore, sodium isostearoyl lactylate is considered to be non-sensitiser in this test.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E, the sensitisation potential of the test item sodium isostearoyl lactylate was assessed on the basis of dendritic cell activation by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1.

Cells were incubated with the test item (8 concentrations; range 40.53-145.24 µg/mL) for 24 h at 37 °C. After exposure cells were labelled with fluorescent antibodies of cell surface markers CD54 and CD86 and the expression levels of CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

The lowest non-cytotoxic (i.e. cell viability of ≥50%) concentration was 100.86 µg/mL. At this concentration the expression levels of CD86 (128% experiment 1, 76% experiment 2) and CD54 (106% experiment 1, 96% experiment 2) were not upregulated above the criteria for a positive test outcome (i.e. 150% for CD86 and 200% for CD54).

Therefore, sodium isostearoyl lactylate is considered as a non-sensitiser in this test.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-08-22 to 2018-11-06

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:

adopted 04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of European Centre for the Validation of Alternative Methods (ECVAM) and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Pationic ISL (Lot: 1731200025)
- Expiration date of the lot/batch: 12 months after opening

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: All test item solutions were freshly prepared immediately prior to use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed in a glass vial and was dissolved in methanol. A solution with a concentration of 100 mM was prepared.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

Controls: Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.

Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was dissolved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

Co-elution controls were set up in parallel to sample preparation but without the respective peptide solutions. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Reference Controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Three replicates of this RC were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Three replicates of this RC were injected in the beginning and in the end of each HPLC run.
Two reference controls C were set up for the test item and for the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent (methanol) used to solubilise the test item. The controls were used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally, reference control C was used to calculate PPD.
The RC Cs with acetonitrile and the test item solvent (methanol) were included in every assay run for both peptides and were injected just before the positive control and the test item samples.

Pre-Experiments
Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated with the following solvents suitable for the test:
- acetonitrile
- dist. water
- dist. water : acetonitrile 1:1 (v/v)
- isopropanol
- methanol
- 1,4-butanediol
- N,N-dimethylformamide
The test item was not soluble in acetonitrile, dist. water, isopropanol and 1,4-butanediol. The test item was completely soluble in dist. water: acetonitrile 1:1 (v/v) (however, a period of 20–30 minutes was required for the test item to be completely dissolved in this solvent), methanol and N,N dimethylformamide. Among these three solvents, methanol was chosen as the suitable vehicle for the main experiments.

Experimental Procedure
Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 for the cysteine peptide, 1:50 for the lysine peptide). The reaction solutions were incubated in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in Table 1 in "Any other information on materials and methods" section. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.

Preparation of the HPLC Standard Calibration Curve
A standard calibration curve was generated for each peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions for each peptide (0, 0.017, 0.033, 0.067, 0.134, 0.267 and 0.534 mM).

HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.

The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

The concentrations of the cysteine and lysine peptide were determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated according to the following formula:

PPD = [1 – (Peptide PA in the replicate injection/mean peptide PA in reference control C)] x 100

The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent, a co-elution was assumed and the evaluation would be adjusted accordingly.

Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive and to be a skin sensitiser if the mean depletion of both peptides exceeds the threshold of prediction model 1 (see Table 2 in "Any other information on materials and methods" section). Negative depletion is considered as “0” when calculating the mean.

By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model), the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin if the mean depletion of both peptides is below 6.38%.

In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide, prediction model 2 can be applied (cysteine 1:10 prediction model) (see Table 3 in "Any other information on materials and methods" section).

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.92% (using prediction model 1).

Key result

Parameter:

other: Mean percent of peptide depletion

Value:

0.85

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

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

For individual results see Table 4 in box "Any other information on results incl. tables".

Table 4:   Depletion of the Cysteine Peptide

Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.2840	0.1493	72.34	72.48	0.20	0.27
	4.2280	0.1473	72.70
	4.2760	0.1490	72.39
Test Item	15.1010	0.5235	0.16	0.80	0.71	89.54
	14.8880	0.5161	1.57
	15.0260	0.5209	0.66

 

Table 5:   Depletion of the Lysine Peptide

Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	5.8160	0.1934	61.85	61.36	0.58	0.95
	5.8670	0.1950	61.51
	5.9890	0.1991	60.71
Test Item	15.3950	0.5113	0.26	0.89	0.59	65.47
	15.2790	0.5074	1.01
	15.2170	0.5054	1.41

 

Table 6: Prediction of sensitising potential of the test item

	Prediction Model (Cysteine 1:10 and Lysine 1:50)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	0.85	Minimal Reactivity	negative
Positive Control	66.92	High Reactivity	positive

Interpretation of results:

GHS criteria not met

Conclusions:

In this study (performed in accordance with OECD guideline 442C) under the given conditions sodium isostearoyl lactylate showed minimal reactivity (as indicated by peptide depletion) towards both cysteine and lysine peptides. Sodium isostearoyl lactylate is thus considered as a non-sensitiser in this test.

Executive summary:

In the in chemico sensitisation study performed in accordance with OECD guideline 442C, the test item sodium isostearoyl lactylate was dissolved in methanol. Based on its molecular weight of 450.59 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by high-performance liquid chromatography (HPLC).

From the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. No co-elution of the test item with the peptide peaks was observed.

Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item-incubated samples to the corresponding reference control C (RC C_methanol).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was 0.85%, which is below the threshold level of 6.38% based on the prediction model using the mean cysteine (1:10) as well as lysine (1:50) percent peptide depletion values. Thus the test item sodium isostearoyl lactylate is considered as a non-sensitiser in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In two in vitro skin sensitisation studies conducted according to OECD 442D and OECD 442E and one in chemico skin sensitisation study conducted according to OECD 442C, sodium isostearoyl lactylate was tested for its skin sensitisation potential.

Based on the results (one test showing sensitising potential, whereas two tests showing no sensitising potential) as a weight-of-evidence approach, no classification of sodium isostearoyl lactylate for skin sensitisation is warranted.