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

Endpoint summary

Description of key information

The skin sensitisation potential of the substance sodium lauroyl lactylate was assessed in two in vitro skin sensitisation assays (OECD 442D and OECD 442E). In the first study conducted according to OECD 442D, the skin sensitisation potential of the target substance was assessed based on the activation of keratinocytes using the in vitro KeratinoSens cell line. In the second study conducted according to OECD 442E, the sensitisation potential of the target substance 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 both studies, sodium lauroyl 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:

2018-03-22 to 2018-11-22

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:

Purity: technically pure

SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Lot No. 1805900121

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature; protected from light
- Solubility and stability of the test substance in the solvent/vehicle: The test item was dissolved in distilled water. A stock solution of 200 mM was prepared for experiment 1 and experiment 2 and a stock solution of 6.25 mM was prepared for experiment 3 by pre-weighing the test material into suitable tubes. A 0.2 µm sterile filter was used to sterilise the test sample solution.

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.

- Final dilution of a dissolved solid, stock liquid or gel: With the stock solutions, serial dilutions were made using the solvent (dist. water) to obtain 12 master concentrations of the test item (0.098 to 200 mM for experiments 1 and 2 and 0.27 to 6.25 mM for experiment 3). The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2 for experiments 1 and 2 and a constant dilution factor of 1:1.33 for experiment 3. 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 incubated with the cells so that the final concentrations of the tested chemical ranged from 0.98 to 2000 µM in experiments 1 and 2 and from 2.71 to 62.50 µM in experiment 3.
Since the test item was dissolved in water and the positive control was dissolved in DMSO, DMSO was added to all test item preparations to a final concentration of 1% (v/v) to allow comparison of the test item concentrations with the positive and negative (solvent) controls.

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 (P) < 25 (P 05 in experiment 1; P 07 in experiment 2; P 06 in experiment 3) were used.

Cells were cultured in 75 cm² culture flasks (Greiner) in maintenance medium at 37 ± 1 °C and 5 % CO2 in a humidified incubator. For test item exposure, cells were cultured in 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 manufacture’s manual.

Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilised)
- 10 × 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 5× Reagent
The kit (Promega, Cat. No.: E1531) consisted of 30 mL Luciferase Cell Culture Lysis 5× Reagent.
Prior to use lysis buffer was diluted 1:5 with distilled water.

DOSE GROUPS:
1. Negative/Solvent Control: 1 % (v/v) DMSO in test item exposure medium
2. Positive Control (cinnamic aldehyde): 4 µM; 8 µM; 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative controls were assessed using six replicates per 96-well plate in every independent run.

EXPERIMENTAL PROCEDURE:
The incubation was performed in 96-well plates. Cells were counted by Neubauer chamber and a cell suspension of 8.0E+04 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1.0E+04 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 (experiment 3) or over the weekend (experiments 1 and 2). After the incubation period the plate was shaken for 10 min and the optical density (OD) was measured at λ = 600 nm using a plate reader (Tecan, Infinite 200Pro).

DATA ANALYSIS:
Cell viability, maximal induction of the luciferase activity (Imax), EC1.5 value (concentration for which induction of luciferase activity is above the 1.5-fold threshold), IC30 and IC50 (concentrations causing 50 % and 30 % reduction of cellular viability) are determined. 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.
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.
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 negative/solvent control,
- cell viability is > 70 % at the lowest concentration with an induction of luciferase activity > 1.5-fold relative to the negative/solvent control,
- 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 (4.34 in experiment 1; 2.07 in experiment 2; 3.97 in experiment 3).
- The calculated EC1.5 was between 7 and 30 µM (16.27 µM in experiment 1; 26.58 µM in experiment 2; 21.01 in experiment 3).

Key result

Run / experiment:

other: 1

Parameter:

other: Lowest tested concentration (µM) with a significant luciferase induction of >1.5-fold

Value:

31.25

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: borderline results

Remarks:

Relative cell viability: 76.0%

Key result

Run / experiment:

other: Experiment 1, at 31.25 µM

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: borderline results / relative cell viability: 76.0%

Key result

Run / experiment:

other: 2

Parameter:

other: Lowest tested concentration (µM) with a significant luciferase induction of >1.5-fold

Value:

15.63

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: borderline results

Remarks:

Relative cell viability: 76.3%

Key result

Run / experiment:

other: Experiment 2, at 15.63 µM

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.56

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: borderline results / relative cell viability: 76.3%

Key result

Run / experiment:

other: 3

Parameter:

other: Lowest tested concentration (µM) with a significant luciferase induction of >1.5-fold

Value:

46.99

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Relative cell viability: 51.7%

Key result

Run / experiment:

other: Experiment 3, at 46.99 µM

Parameter:

other: max. luciferase activity (Imax) induction

Value:

1.99

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Relative cell viability: 51.7%

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed.
- For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p < 0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.
- The lowest concentration with > 1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30 % reduction on cellular viability at the EC1.5 determining concentration.

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

For the individual and overall results of the 3 experiments see Tables 1-5 in box 'Any other information on results incl. tables'.

Table 1: Results of the Cytotoxicity Measurement

	Conc. [µM]	Relative Cell Viability [%]^1				Experiment 3
		Experiment 1	Experiment 2	Mean	SD	Conc. [µM]	Relative Cell Viability [%]^1
Negative/ Solvent Control	-	100	100	100	0.0	-	100
Positive Control	4.00	101.3	92.7	97.0	6.1	4.00	93.4
	8.00	100.5	94.2	97.4	4.5	8.00	101.4
	16.00	98.8	93.7	96.2	3.6	16.00	104.9
	32.00	101.8	95.8	98.8	4.2	32.00	100.5
	64.00	96.8	91.6	94.2	3.6	64.00	91.5
Test Item	0.98	103.1	141.3	122.2	27.0	2.71	84.4
	1.95	106.2	96.2	101.2	7.1	3.61	89.5
	3.91	102.5	95.7	99.1	4.8	4.80	96.7
	7.81	100.3	91.8	96.1	6.0	6.38	99.6
	15.63	85.3	76.3	80.8	6.4	8.49	108.4
	31.25	76.0	55.9	65.9	14.2	11.29	108.1
	62.50	26.3	0.4	13.4	18.4	15.02	96.1
	125.00	0.1	0.1	0.1	0.0	19.97	98.8
	250.00	0.0	0.2	0.1	0.1	26.57	80.7
	500.00	0.0	0.2	0.1	0.1	35.33	79.4
	1000.00	0.4	0.1	0.3	0.2	46.99	51.7
	2000.00	0.0	0.1	0.1	0.1	62.50	47.8

¹Percentage of cell viability is relative to the solvent control (i.e.set at 100%); Conc.: concentration

 

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Relative Fold Induction^1					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Negative/ Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.14	1.06	0.99	1.07	0.07
	8.00	1.20	1.39	1.54	1.37	0.17
	16.00	1.28	1.59	1.57	1.48	0.18
	32.00	2.36	2.74	2.58	2.56	0.19	*
	64.00	4.04	5.02	3.97	4.34	0.58	*
Test Item	0.98	0.87	1.03	0.95	0.95	0.08
	1.95	0.77	0.85	0.77	0.79	0.05
	3.91	0.87	0.78	0.81	0.82	0.05
	7.81	0.88	0.84	0.79	0.84	0.04
	15.63	1.15	1.13	1.10	1.13	0.02
	31.25	1.72	1.77	1.60	1.70	0.09	*
	62.50	8.96	9.71	7.54	8.74	1.10	*
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

 

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Relative Fold Induction^1					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Negative/ Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.09	1.11	1.16	1.12	0.04
	8.00	1.10	1.28	1.13	1.17	0.10
	16.00	1.19	1.27	1.25	1.23	0.04
	32.00	1.40	2.06	1.44	1.64	0.37
	64.00	1.87	2.02	2.32	2.07	0.23	*
Test Item	0.98	0.65	0.76	1.01	0.81	0.18
	1.95	0.86	1.46	1.30	1.21	0.31
	3.91	1.03	1.30	1.40	1.24	0.19
	7.81	1.04	1.46	1.14	1.22	0.22
	15.63	1.37	1.79	1.50	1.56	0.21	*
	31.25	1.24	1.54	1.67	1.48	0.22
	62.50	1.27	0.77	3.94	1.99	1.70
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

 

Table 4: Induction of Luciferase Activity Experiment 3

Experiment 3	Concentration [µM]	Relative Fold Induction^1					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Negative/ Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.06	1.04	1.06	1.05	0.01
	8.00	1.25	1.46	1.20	1.30	0.14
	16.00	1.39	1.48	1.18	1.35	0.16
	32.00	2.03	1.76	1.71	1.83	0.17	*
	64.00	4.12	4.41	3.38	3.97	0.53	*
Test Item	2.71	1.29	1.25	1.10	1.22	0.10
	3.61	1.03	1.40	1.05	1.16	0.21
	4.80	1.13	1.24	1.22	1.20	0.06
	6.38	1.06	1.10	1.16	1.11	0.05
	8.49	1.21	1.16	1.15	1.17	0.03
	11.29	1.32	1.22	1.08	1.21	0.12
	15.02	1.28	1.41	1.24	1.31	0.09
	19.97	1.19	1.24	0.93	1.12	0.17
	26.57	1.45	1.55	1.05	1.35	0.26
	35.33	1.32	1.48	1.19	1.33	0.15
	46.99	1.92	2.30	1.74	1.99	0.29	*
	62.50	4.33	4.72	3.80	4.28	0.46	*

* = significant induction according to Student’s t-test, p<0.05; Rep.: Replicate

¹Percentage of fold induction is relative to the negative/ solvent control (i.e.set at 1.0).

 

Table 5: Induction of Luciferase Activity – Overall Induction

Overall Induction	Conc. [µM]	Relative Fold Induction^1				Exp. 3
		Exp. 1	Exp. 2	Mean	SD	Conc. [µM]	Relative Fold Induction^1
Negative/ Solvent Control	-	1.00	1.00	1.00	0.00	-	1.00
Positive Control	4.00	1.07	1.12	1.09	0.04	4.00	1.05
	8.00	1.37	1.17	1.27	0.14	8.00	1.30
	16.00	1.48	1.23	1.36	0.17	16.00	1.35
	32.00	2.56	1.64	2.10	0.65	32.00	1.83
	64.00	4.34	2.07	3.21	1.61	64.00	3.97
Test Item	0.98	0.95	0.81	0.88	0.10	2.71	1.22
	1.95	0.79	1.21	1.00	0.29	3.61	1.16
	3.91	0.82	1.24	1.03	0.30	4.80	1.20
	7.81	0.84	1.22	1.03	0.27	6.38	1.11
	15.63	1.13	1.56	1.34	0.30	8.49	1.17
	31.25	1.70	1.48	1.59	0.15	11.29	1.21
	62.50	8.74	1.99	5.37	4.77	15.02	1.31
	125.00	0.00	0.00	0.00	0.00	19.97	1.12
	250.00	0.00	0.00	0.00	0.00	26.57	1.35
	500.00	0.00	0.00	0.00	0.00	35.33	1.33
	1000.00	0.00	0.00	0.00	0.00	46.99	1.99
	2000.00	0.00	0.00	0.00	0.00	62.50	4.28

¹Percentage of fold induction is relative to the negative/ solvent control (i.e.set at 1.0).

Exp. = Experiment, conc. = concentration

Interpretation of results:

GHS criteria not met

Conclusions:

In this study (performed in accordance with OECD 442D) under the given conditions the observed induction of luciferase activity was triggered mainly by the cytotoxicity of the test item sodium lauroyl lactylate rather than by a sensitisation mechanism. Therefore, the test item is considered as a non-sensitiser in this study.

Executive summary:

In a skin sensitisation study conducted according to OECD 442D, the sensitisation potential of the test item sodium lauroyl lactylate (technically pure) in distilled water 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. Cell viability was also assessed by measuring the reduction of MTT.

In the first experiment, a max luciferase activity induction (Imax ± standard deviation/SD) of 8.74 ± 1.10 was determined at a test item concentration of 62.50 µM. However, at this concentration, the relative cell viability was < 70 % (26.3 %) and thus the concentration was considered as cytotoxic. The lowest tested concentration with a significant luciferase induction of > 1.5-fold (1.70 ± SD 0.09) was found to be 31.25 µM. The corresponding relative cell viability was > 70 % (76.0 %). The calculated EC_1.5 was < 1000 µM (25.84 µM). No clear dose-response relationship for luciferase activity induction was observed in the non-cytotoxic concentration range.

In the second experiment, a max luciferase activity induction (Imax ± SD) of 1.99 ± 1.70 was determined at a test item concentration of 62.50 µM. Similar to the first experiment, the relative cell viability at this concentration was < 70 % (0.4 %) and thus the concentration was considered as cytotoxic. The lowest tested concentration with a significant luciferase induction of > 1.5-fold (1.56 ± SD 0.21) was found to be 15.63 µM. The corresponding relative cell viability was > 70 % (76.3 %). However, with a SD of ± 0.21 µM the fold induction value of 1.56 can be considered as borderline result. Furthermore, the concentration of 31.25 µM was considered cytotoxic (relative cell viability of 55.9 %) and had a luciferase activity fold induction of 1.48, which did not meet criterion of 1.5-fold induction for a positive outcome. The calculated EC_1.5 was < 1000 µM (14.34 µM). No clear dose-response relationship for luciferase activity induction was observed in the non-cytotoxic concentration range.

Due to the inconclusive results as well as the induction of the luciferase activity being very close to the cytotoxic levels in both experiments 1 and 2, a third experiment with a narrower concentration range was performed.

In the third experiment, a max luciferase activity induction (Imax ± SD) of 4.28 ± 0.46 was determined at a test item concentration of 62.50 µM. At this concentration, the relative cell viability was < 70 % (47.8 %) and thus the concentration was considered as cytotoxic. The lowest tested concentration with a significant luciferase induction of > 1.5-fold (1.99 ± SD 0.29) was found to be 46.99 µM. The corresponding relative cell viability was < 70 % (51.7 %), which was also considered as cytotoxic. The calculated EC_1.5 was < 1000 µM (38.33 µM). No clear dose-response relationship for luciferase activity induction was observed in the non-cytotoxic concentration range. In this third experiment the clear correlation between cytotoxicity and luciferase induction was shown. At the concentration of 35.33 µM, no cytotoxicity (relative cell viability of 79.4 %) and no significant increase in luciferase activity (fold induction of 1.33) was observed, whereas at the next higher concentration of 46.99 µM, cytotoxicity (relative cell viability of 51.7 %) and significant increase in luciferase activity (fold induction of 1.99) was observed.

Therefore, it can be concluded that the observed induction of luciferase activity was mainly triggered by the cytotoxicity of the test item rather than by a sensitisation mechanism. Under the conditions of this study sodium lauroyl lactylate is considered as a non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-22 to 2018-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation 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:

- Purity: technically pure

SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Esterlac SLL (Lot No. 1805900121)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature; protected from light
- 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: Sonication and warming to 50 °C for 10-20 minutes were performed to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration 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 then prepared by diluting each stock solution 250 times with 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 0.1–0.2E+06 cells/mL.
Cells were cultured in 75 cm² 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 % foetal 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 0.1–0.2E+06 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 2.0E+06 cells/mL. Then 500 µL of the cell suspension were seeded onto a 24 well flat-bottom plate (1.0E+06 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 × 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 0.1–0.2E+06 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 × g) and were re-suspended in fresh culture medium at a density of 2.0E+06 cells/mL. Then 500 µL of the cell suspension were seeded onto a 24 well flat-bottom plate (1.0E+06 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 × 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 × 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 × CV75 is < 90 %. In contrast, a positive test outcome was accepted irrespective of cell viability of > 90 % at a concentration of 1.2 × 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 (302 % experiment 1; 275 % experiment 2) and 200 % for CD54 (428 % experiment 1; 248 % experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: CD86 upregulation

Remarks:

at highest tested concentration of 125.56 µg/mL

Value:

2 273

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

severe cytotoxicity

Key result

Run / experiment:

other: 1

Parameter:

other: CD54 upregulation

Remarks:

at highest tested concentration of 125.56 µg/mL

Value:

3 499

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

severe cytotoxicity

Key result

Run / experiment:

other: 2

Parameter:

other: CD86 upregulation

Remarks:

at highest tested concentration of 125.56 µg/mL

Value:

102

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

severe cytotoxicity

Key result

Run / experiment:

other: 2

Parameter:

other: CD54 upregulation

Remarks:

at highest tested concentration of 125.56 µg/mL

Value:

94

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

severe cytotoxicity

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 Tables 1 and 2 in box "Any other information on results incl. tables".

Summary of Results:

Severe cytotoxicity was observed for the cells treated with the test item. Relative cell viability at the highest test item concentration of 125.56 μg/mL was reduced to 7.6 % (CD86), 7.5 % (CD54) and 7.8 % (isotype IgG1 control) compared to the solvent control in the first experiment and to 13.4 % (CD86), 13.3% (CD54) and 12.4 % (isotype IgG1 control) compared to the solvent control in the second experiment.

In both experiments, the expression of the cell surface marker CD86 was upregulated above the threshold of 150 % relative to the solvent control at the highest test item concentration of 125.56 μg/mL (2273 % in the first experiment, 203 % in the second experiment). At this concentration the test item showed severe cytotoxicity. No further upregulation above the threshold of 150 % was observed for CD86 in the other tested concentrations.

In both experiments, the expression of the cell surface marker CD54 was upregulated above the threshold of 200 % relative to the solvent control at the highest test item concentration of 125.56 μg/mL (3499 % in the first experiment, 377 % in the second experiment). At this concentration the test item showed severe cytotoxicity. No further upregulation above the threshold of 200 % was observed for CD54 in the other tested concentrations.

Table1: CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Mean Fluorescence Intensity (corrected for IgG1)		Relative Fluorescence Intensity (RFI) [%]^1		Cell marker to Isotype IgG1 [%]^2
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	94.7	95.4	95.2	2174	1124	610	1564	514	84	95	356	184
Solvent Control (0.2% THF)	-	94.8	94.4	94.9	2036.0	1190.0	637.0	1399	553	100	100	320	187
Solvent Control (0.2% DMSO)	-	94.0	94.3	94.1	2469	1137	597	1872	540	100	100	414	190
DNCB	4.00	77.2	76.8	77.1	6275	2930	617	5658	2313	302	428	1017	475
Sodium lauroyl lactylate	125.56	7.6	7.5	7.8	34370	21922	2574	31796	19348	2273	3499	1335	852
	104.63	14.0	14.5	14.2	2363	1698	795	1568	903	112	163	297	214
	87.19	85.3	84.1	84.3	2473	1316	695	1778	621	127	112	356	189
	72.66	90.9	91.4	89.7	2403	1248	705	1698	543	121	98	341	177
	60.55	92.6	92.7	91.5	2317	1168	709	1608	459	115	83	327	165
	50.46	93.5	93.1	93.2	2234	1220	695	1539	525	110	95	321	176
	42.05	93.5	94.3	93.6	2453	1216	700	1753	516	125	93	350	174
	35.04	93.7	94.1	93.3	2259	1179	677	1582	502	113	91	334	174

 

Table2:  CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Mean Fluorescence Intensity (corrected for IgG1)		Relative Fluorescence Intensity (RFI) [%]^1		Cell marker to Isotype IgG1 [%]^2
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	92.0	93.2	92.6	3138	1369	616	2522	753	98	109	509	222
Solvent Control 2 (0.2% THF)	-	93.4	93.7	93.6	2849	1258	672	2177	586	100	100	424	187
Solvent Control 1 (0.2% DMSO)	-	93.5	93.2	93.4	3159	1280	590	2569	690	100	100	535	217
DNCB	4.0	76.9	77.2	76.9	7652	2300	586	7066	1714	275	248	1306	392
Sodium lauroyl lactylate	125.56	13.4	13.3	12.4	5733	3528	1319	4414	2209	203	377	435	267
	104.63	28.8	29.6	29.7	3071	1607	743	2328	864	107	147	413	216
	87.19	70.5	69.1	69.7	3288	1466	735	2553	731	117	125	447	199
	72.66	88.6	89.2	89.0	3024	1392	765	2259	627	104	107	395	182
	60.55	90.9	90.7	90.7	3250	1334	772	2478	562	114	96	421	173
	50.46	91.0	91.9	92.1	2983	1327	781	2202	546	101	93	382	170
	42.05	92.2	92.6	91.7	3033	1337	734	2299	603	106	103	413	182
	35.04	93.2	93.7	93.4	2984	1301	753	2231	548	102	94	396	173

 

RFI is relative to the respective solvent control (i.e. 0.2% DMSO for DNCB and medium control and 0.2% THF for the test item).

¹(MFI_{negative control, solvent control, positive control, test item}* 100) / MFI_{solvent control}(all corrected for IgG1)

²(MFI_{negative control, solvent control, positive control, test item}* MFI_{solvent control}) * 100

 

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 lauroyl lactylate did not trigger an upregulation of the expression of the cell surface markers CD86 and CD54 in two independent experiment runs. Therefore, the test item 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 lauroyl lactylate (technically pure) in tetrahydrofuran 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 35.04–125.56 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.

In both experiments, the expression of the cell surface marker CD86 was upregulated above the threshold of 150 % relative to the solvent control at the highest test item concentration of 125.56 µg/mL (2273 % in the first experiment, 203 % in the second experiment). At this concentration the test item showed severe cytotoxicity. No further upregulation above the threshold of 150 % was observed for CD86 in the other tested concentrations.

In both experiments, the expression of the cell surface marker CD54 was upregulated above the threshold of 200 % relative to the solvent control at the highest test item concentration of 125.56 µg/mL (3499 % in the first experiment, 377 % in the second experiment). At this concentration the test item showed severe cytotoxicity. No further upregulation above the threshold of 200 % was observed for CD54 in the other tested concentrations.

Therefore, sodium lauroyl lactylate is considered to be a non-sensitiser in this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential of the substance sodium lauroyl lactylate was assessed in two in vitro skin sensitisation assays (OECD 442D and OECD 442E). In the first study conducted according to OECD 442D, the skin sensitisation potential of the target substance was assessed based on the activation of keratinocytes using the in vitro KeratinoSens cell line. In the second study conducted according to OECD 442E, the sensitisation potential of the target substance 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 both studies, sodium lauroyl lactylate is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Sodium lauroyl lactylate was tested for its skin sensitisation potential in two in vitro skin sensitisation studies conducted according to OECD 442D and OECD 442E. Based on the results of both studies, classification of sodium lauroyl lactylate for skin sensitisation is not warranted.