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

Endpoint summary

Description of key information

The potential of sodium lauroyl lactylate (target substance) to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). Moreover, suitable in vivo test data is available showing that the target substance as a 10% concentrated solution did not induce any effects on the skin or eye of rabbits. An eye irritation study did not need to be conducted in accordance with REACH Regulation (EC) No 1907/2006, Annex VII, column 2 of information requirement section 8.2. Based on the results, sodium lauroyl lactylate must be considered as corrosive to skin and eye and is therefore classified as Skin Corr. 1A, H314 and Eye Dam. 1, H318 with a specific concentration limit of ≤ 10% to not induce any effects.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-11-14 to 2019-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name: Sodium lauroyl lactylate
- Chemical Name: Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate
- CAS No.: 13557-75-0
- Batch No.: 1829200025
- Molecular Weight: 366.425 g/mol
- Physical State at Room Temperature: solid
- Colour: light yellow
- Storage Conditions: room temperature, protected from light
- Stability: undergoes hydrolysis in water
- Expiry Date: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Sterile DPBS (25 µL) was first applied to the EpiDerm skin tissue surface in order to improve the contact between the test item and the epidermis. As the test item was very viscous, it was not possible to apply the test item directly on the skin tissue surface. Thus, a nylon mesh was used as application aid. Before using a nylon mesh, mesh compatibility of the test item was tested to exclude possible interactions of the test item with the nylon mesh that might result in disintegration of the mesh. Microscopic examination of the nylon mesh treated for 60 min with 25 mg of the test item showed no interactions. The test item (25 mg; 39 mg/cm²) were applied onto a nylon mesh using an application spoon. This nylon mesh was then placed upside down atop the EpiDerm tissue. Due to the stickiness of the test item, it was not possible to evenly distribute the test item on the nylon mesh. However, the surface of the tested tissue samples could be entirely covered with the test item during the main experiment, so the outcomes of this study are not affected by the physical state of the test item.

Test system:

human skin model

Remarks:

EpiDerm

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 28672

EpiDerm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
- 1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 28672)
- 2x 24-well plates
- 8x 6-well plates
- 1x bottle of assay medium (DMEM-based medium, Lot No.: 112918MSD)
- 1x bottle of DPBS Rinse Solution (Lot No.: 0710108MSA)
- 1x 1 vial 5% SDS Solution (TC-SDS-5%)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for the first 35 ± 1 min, afterwards the plates were placed under the sterile flow until 60 ± 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1996962)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS solution (TC-SDS 5%, MatTek, CAS No.: 151-21-3, Lot No: 022118ISA).

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 ± 4 h

Number of replicates:

3 tissues per dose group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

13.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

For detailed results see box "Any other information on results incl. tables".

Pre-experiments:

The solution of 25 mg of the test item per 1 mL MTT medium showed no reduction of MTT compared to the MTT medium alone. The solution did not turn blue/purple. There was no non-specific reduction of MTT (NSMTT) by the test item, i.e. NSMTT equalled 0%.

The solution of 25 mg of the test item per 300 µL aqua dest. or 300 µL isopropanol also showed no colouring that was detectable by unaided eye assessment. There was no non-specific colouring (NSC) by the test item, i.e. NSC equalled 0%. Overall, the test item showed no non-specific reduction of MTT and no relevant colouring when mixed with aqua dest. or isopropanol. Therefore, no additional controls for correction of possible false negative results were necessary for the main experiment. As the test item was very viscous, it was not possible to apply the test item directly on the skin tissue surface. Thus, a nylon mesh was used as application aid. Microscopic examination of the nylon mesh in contact with 25 mg of the test item for 60 min showed no interactions. Due to the stickiness of the test item, it was not possible to evenly distribute the test item on the nylon mesh. However, the surface of the tested tissue samples could be entirely covered with the test item during the main experiment, so the outcomes of this study are not affected by the physical state of the test item.

Results of the main experiment:

Table 1: Results of the main experiment

Name	Negative Control****			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.441	1.529	1.420	0.284	0.251	0.221	0.222	0.227	0.230
	1.445	1.546	1.420	0.294	0.260	0.225	0.230	0.225	0.237
OD570 (Blank Corrected)	1.400	1.488	1.379	0.244	0.210	0.181	0.182	0.186	0.189
	1.404	1.506	1.379	0.254	0.220	0.184	0.190	0.185	0.197
Mean OD570 of the Duplicates (Blank Corrected)	1.402	1.497	1.379	0.249	0.215	0.183	0.186	0.185	0.193
Total Mean OD570 of 3 Replicate Tissues (Blank Corrected)	1.426*			0.215			0.188
SD of Mean OD570 of 3 Replicate Tissues (Blank Corrected)	0.062			0.033			0.004
Relative Tissue Viability [%]	98.3	105.0	96.7	17.4	15.1	12.8	13.0	13.0	13.5
Mean Relative Tissue Viability [%]	100.0			15.1**			13.2
SD of Relative Tissue Viability [%]***	4.4			2.3			0.3
CV of Relative Tissue Viability [%]	4.4			15.3			2.3

* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is ≤ 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

****The mean absolute OD_{570 nm} of the negative control is 1.467, which fulfils the test acceptance criteria (mean absolute OD_570nmof the three negative control tissues is ≥ 0.8 and ≤ 2.8).

Table 2: Quality criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NC	1.467	0.8 ≤ NC ≤ 2.8	pass
Relative Viability [%] PC	15.1	≤ 20%	pass
SD of Relative Viability [%] (min-max)	0.3 -4.4	≤ 18%	pass

NC: Negative Control

PC: Positive Control

Interpretation of results:

other: Considered to be an irritant to the skin (UN GHS Category 1 or 2). Further testing is required in order to distinguish between these two categories.

Conclusions:

In conclusion, based on the results obtained from this in vitro skin irritation study (OECD 439), sodium lauroyl lactylate is considered to be an irritant to the skin (UN GHS Category 1 or 2). Further testing is required in order to distinguish between these two categories.

Executive summary:

In an in vitro dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to to the test item sodium lauroyl lactylate for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability of the test item (% negative control) was ≤ 50% (13.2%). Based on this result, the test item sodium lauroyl lactylate is considered to be irritating to the skin in accordance with UN GHS "Category 1 or 2" and further testing is required in order to distinguish between these two categories.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

other: Industry bulletin report

Adequacy of study:

supporting study

Study period:

1979

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

other: Regulations for the Enforcement of the Federal Hazardous Substances Act

Version / remarks:

17 September 1964

Principles of method if other than guideline:

Primary skin irritation & corrosivity in albino rabbits (evaluated in accordance with the techniques specified in the Regulations for the Enforcement of the Federal Hazardous Substances Act (Revised Federal Register September 17, 1964))

GLP compliance:

not specified

Specific details on test material used for the study:

- Test material name: Pationic 138C
- Final concentration: 10 % (w/w)

Species:

rabbit

Strain:

other: Albino

Amount / concentration applied:

10% (w/w) concentration

Irritation parameter:

primary dermal irritation index (PDII)

Score:

0

Remarks on result:

no indication of irritation

Interpretation of results:

study cannot be used for classification

Conclusions:

Sodium lauroyl lactylate (Pationic 138C; 10 % concentration) has been shown to have no skin irritation potential in albino rabbits.

Executive summary:

The bulletin by Patco Cosmetic Products showed no indication of skin irritation (primary irritation index of 0) in albino rabbits for the test item sodium lauroyl lactylate (Pationic 138C; 10 % concentration). It was mentioned in the paper that the study was evaluated in accordance with the techniques specified in the Regulations for the Enforcement of the Federal Hazardous Substances Act (Revised Federal Register September 17, 1964).

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-04-05 to 2019-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name: Sodium lauroyl lactylate
- Chemical Name: Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate
- CAS No.: 13557-75-0
- Batch No.: 1829200025
- Appearance: solid (viscous) at room temperature
- Colour: light yellow
- Storage conditions: room temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was solid enough at room temperature to be ground to a powder in a mortar with pestle. 25 mg of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 µL H2O. The test item was spread to match size of the tissue.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 28696
EpiDerm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
- 1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 28696)
- 2x 24-well plates
- 4x 6-well plates
- 1x bottle of assay medium (DMEM-based medium, Lot No.: 050919MSB)
- 1x bottle of DPBS Rinse Solution (Lot No.: 031219MSA)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

FURTHER REAGENTS
- MTT stock solution: 5 mg/mL MTT (VWR; Lot 18I1156332) in PBS (Gibco; Lot No.: 2026787)
- MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Isopropanol (AppliChem; Lot No.: 0001365249)
- Aqua dest. (Sigma Aldrich; Lot No.: RNBG3520)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (MTT incubation): 37 ± 1 °C (for 3 h, 5.0% CO2 / 95% air)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
At the end of exposure each tissue was rinsed about 20 times with PBS by filling and emptying the tissue insert. Excess liquid was carefully removed and transferred into new wells pre-filled with 0.3 mL/well pre-warmed MTT solution.
After 3 h MTT incubation tissues were rinsed twice in PBS and dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL/well of MTT ready-to-use solution
- Incubation time: 3 h min at 37 ± 1 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL H2O

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8N KOH

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

two

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes /mean of two replicates

Value:

97.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes / mean of two replicates

Value:

4.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

For detailed results please refer to box "Any other information on results incl. tables".

OTHER EFFECTS:
Pre-experiments:
The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT is determined to be 0%. The mixture of 25 mg test item per 300 µL Aqua dest. and per 300 µL isopropanol showed no colouring as compared to the solvent. Therefore, NSC is determined to be 0%. The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with Aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

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

Table 1: Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NC (3 min Experiment)	1.708	0.8 ≤ NC ≤ 2.8	pass
Mean Absolute OD570 nm NC (60 min Experiment)	1.902	0.8 ≤ NC ≤ 2.8	pass
Mean Relative Tissue Viability [%] of PC (60 min experiment)	3.7	< 15%	pass
CV [%] (in the range of 20 – 100% viability)	1.3 – 6.4	≤ 30%	pass

NC: negative control

PC: positive control

Table 2: Results of 3 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.776	1.620	0.101	0.130	1.617	1.689
	1.756	1.630	0.107	0.136	1.603	1.705
	1.817	1.648	0.104	0.132	1.633	1.720
Mean Absolute OD570	1.708****		0.118		1.661
OD570- Blank Corrected	1.730	1.574	0.055	0.084	1.571	1.643
	1.710	1.584	0.061	0.090	1.557	1.659
	1.771	1.602	0.058	0.086	1.587	1.674
Mean OD570of 3 Aliquots (Blank Corrected)	1.737	1.587	0.058	0.087	1.572	1.658
SD OD570 of 3 Aliquots	0.031	0.014	0.003	0.003	0.015	0.016
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.662*		0.072		1.615
SD OD570 of 2 Replicate Tissues	0.106		0.020		0.061
Mean Relative Tissue Viability [%]	100.0		4.3		97.2
Coefficient Of Variation [%]***	6.4		28.3		3.8

* corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

****The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8

Table 3: Results of 60 min Experiment

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.974	1.857	0.095	0.134	0.118	0.124
	1.896	1.875	0.090	0.131	0.119	0.123
	1.886	1.922	0.096	0.138	0.122	0.124
Mean Absolute OD570	1.902****		0.114		0.122
OD570- Blank Corrected	1.928	1.811	0.049	0.088	0.072	0.078
	1.850	1.829	0.044	0.085	0.073	0.077
	1.840	1.876	0.050	0.092	0.076	0.078
Mean OD570 of 3 Aliquots (Blank Corrected)	1.873	1.839	0.048	0.089	0.074	0.078
SD OD570 of 3 Aliquots	0.048	0.034	0.003	0.004	0.002	0.001
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.856*		0.068		0.076
SD OD570 of 2 Replicate Tissues	0.024		0.029		0.003
Mean Relative Tissue Viability [%]	100.0		3.7**		4.1
Coefficient Of Variation [%]***	1.3		42.6		3.6

* corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control < 15%

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

**** The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In conclusion, based on the results obtained from this in vitro skin corrosion study (OECD 431), sodium lauroyl lactylate is considered to be corrosive (UN GHS Category 1B or 1C).

Executive summary:

In an in vitro skin corrosion study conducted according to OECD guideline 431, the test item Sodium lauroyl lactylate was applied topically to the EpiDerm^TM tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues. The mean relative tissue viability (% negative control) was reduced below 15% (4.1%) after 60 min treatment, but not below 50% (97.2%) after 3 min treatment. The positive and negative controls confirmed the validity of the study. Based on the results, the test item should therefore be classified as corrosive (a combination of optional sub-categories 1B and 1C in accordance with UN GHS). 

Reference 4

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-09-26 to 2019-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Chemical Name: Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl laurate
- Product Name: Esterlac SLL Balance+
- Batch No.: PG20192001
- CAS No.: 13557-75-0
- Physical State at RT: solid, very viscous
- Colour: light yellow
- Storage Conditions: room temperature, protection from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was heated to 50 °C under stirring to homogenise the sample. Afterwards it was placed in a water bath to cool the temperature down to 37 °C. The test item was applied undiluted. 50 μL of the test item were dispensed directly atop the EpiDerm tissue. The test item was spread to match size of the tissue.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 28696
EpiDerm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
- 1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 30832)
- 2x 24-well plates
- 4x 6-well plates
- 1x bottle of assay medium (DMEM-based medium, Lot No.: 101719MJB)
- 1x bottle of DPBS Rinse Solution (Lot No.: 092419MSA)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

FURTHER REAGENTS
- MTT stock solution: 5 mg/mL MTT (VWR; Lot 18I1156332 [main exp.], 18I51156 [pre-test]) in PBS (Gibco; Lot No.: 2098592 [main exp.], 2026787 [pre-test])
- MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
- Isopropanol (AppliChem; Lot No.: 0001245299)
- Aqua dest. (Sigma Aldrich; Lot No.: RNBG3520)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (MTT incubation): 37 ± 1 °C (for 3 h, 5.0% CO2 / 95% air)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
At the end of exposure each tissue was rinsed about 20 times with PBS by filling and emptying the tissue insert. Excess liquid was carefully removed and transferred into new wells pre-filled with 0.3 mL/well pre-warmed MTT solution.
After 3 h MTT incubation tissues were rinsed twice in PBS and dried. Then the inserts were transferred into 12-well “extraction plates“. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out either over night without shaking at room temperature or, alternatively, at least 2 h with shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 min. Per each tissue 3 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL/well of MTT ready-to-use solution
- Incubation time: 3 h min at 37 ± 1 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8N KOH

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

two

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes / mean of two replicates

Value:

11.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes / mean of two replicates

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

For detailed results please refer to box "Any other information on results incl. tables".

OTHER EFFECTS:
Pre-experiments:
The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT is determined to be 0%. The mixture of 50 µL test item per 300 µL Aqua dest. and per 300 µL isopropanol showed no colouring as compared to the solvent. Therefore, NSC is determined to be 0%. The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with Aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

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

Table 1: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NC (3 min Experiment)	1.814	0.8 ≤ NC ≤ 2.8	pass
Mean Absolute OD570 nm NC (60 min Experiment)	1.756	0.8 ≤ NC ≤ 2.8	pass
Mean Relative Tissue Viability [%] of PC (60 min experiment)	4.1	≤ 15%	pass
CV [%] (in the range of 20 – 100% viability)	1.8 - 5.8	≤ 30%	pass

Table 2: Results of 3 min Experiment

	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.805	1.785	0.213	0.232	0.261	0.271
	1.845	1.792	0.215	0.235	0.222	0.269
	1.860	1.800	0.221	0.234	0.267	0.268
Mean Absolute OD570	1.814****		0.225		0.260
OD570 - Blank Corrected	1.755	1.735	0.163	0.182	0.211	0.221
	1.795	1.743	0.165	0.185	0.172	0.219
	1.811	1.750	0.171	0.184	0.217	0.219
Mean OD570 of 3 Aliquots (Blank Corrected)	1.787	1.742	0.166	0.184	0.200	0.220
SD OD570 of 3 Aliquots	0.028	0.008	0.004	0.002	0.024	0.002
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.765*		0.175		0.210
SD OD570 of 2 Replicate Tissues	0.032		0.012		0.014
Mean Relative Tissue Viability [%]	100.0		9.9		11.9
Coefficient Of Variation [%]***	1.8		7.1		6.6

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

**** The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8

Table 3: Results of 60 min Experiment

	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.690	1.799	0.122	0.111	0.086	0.090
	1.687	1.830	0.120	0.110	0.086	0.087
	1.679	1.850	0.134	0.122	0.099	0.099
Mean Absolute OD570	1.756****		0.120		0.091
OD570 - Blank Corrected	1.640	1.749	0.072	0.061	0.036	0.040
	1.637	1.780	0.070	0.060	0.037	0.037
	1.630	1.800	0.084	0.072	0.049	0.049
Mean OD570 of 3 Aliquots (Blank Corrected)	1.635	1.776	0.076	0.064	0.041	0.042
SD OD570 of 3 Aliquots	0.005	0.026	0.008	0.007	0.007	0.006
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected)	1.706*		0.070		0.041
SD OD570 of 2 Replicate Tissues	0.100		0.008		0.001
Mean Relative Tissue Viability [%]	100.0		4.1**		2.4
Coefficient Of Variation [%]***	5.8		11.3		2.2

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the 60 min positive control is ≤ 15%,

*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.

**** The mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.8

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Conclusions:

In conclusion, based on the results obtained from this in vitro skin corrosion study (OECD 431), sodium lauroyl lactylate is considered to be corrosive (UN GHS Category 1A).

Executive summary:

In an in vitro skin corrosion study conducted according to OECD guideline 431, the test item Sodium lauroyl lactylate was applied topically to the EpiDerm^TM tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues. The mean relative tissue viability (% negative control) was reduced below 15% (11.9%) after 3 min treatment and below 15% (2.4%) after 60 min treatment. The positive and negative controls confirmed the validity of the study. Based on the results, the test item should therefore be classified as corrosive in accordance with CLP regulation 1272/2008 (Skin Corr 1A, H314). 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

other: Industry bulletin report

Adequacy of study:

supporting study

Study period:

1979

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

other: Regulations for the Enforcement of the Federal Hazardous Substances Act

Version / remarks:

17 September 1964

Principles of method if other than guideline:

Acute Eye Irritation in Albino Rabbits (Evaluated in accordance with the techniques specified in the Regulations for the Enforcement of the Federal Hazardous Substances Act (Revised Federal Register September 17, 1964))

GLP compliance:

not specified

Specific details on test material used for the study:

- Test material name: Pationic 138C
- Final concentration: 10% (w/w)

Species:

rabbit

Strain:

other: Albino

Amount / concentration applied:

10% (w/w) concentration

Remarks on result:

no indication of irritation

Interpretation of results:

study cannot be used for classification

Conclusions:

Sodium lauroyl lactylate (Pationic 138C; 10% concentration) has been shown to have no eye irritation potential in albino rabbits.

Executive summary:

The bulletin paper from Patco showed no indication of eye irritation in albino rabbits for test item sodium lauroyl lactylate (Pationic 138C; 10% concentration). It was mentioned in the paper that the study was evaluated in accordance with the techniques specified in the Regulations for the Enforcement of the Federal Hazardous Substances Act (Revised Federal Register September 17, 1964).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of sodium lauroyl lactylate (target substance) to induce skin irritation/corrosion was evaluated in two suitable in vitro test methods (OECD 439 and OECD 431). Moreover, suitable in vivo test data is available.

In an in vitro dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to the test item sodium lauroyl lactylate for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability of the test item (% negative control) was ≤ 50% (13.2%). Based on this result, the test item sodium lauroyl lactylate is considered to be irritating to the skin in accordance with UN GHS "Category 1 or 2" and further testing is required in order to distinguish between these two categories.

In a first in vitro skin corrosion study conducted according to OECD guideline 431, the test item sodium lauroyl lactylate was applied topically to the EpiDerm^TM tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues. The mean relative tissue viability (% negative control) was reduced below 15% (4.1%) after 60 min treatment, but not below 50% (97.2%) after 3 min treatment. Based on the results, the test item should therefore be classified as corrosive (a combination of optional sub-categories 1B and 1C in accordance with UN GHS).

In the second in vitro skin corrosion study conducted according to OECD guideline 431, the test item sodium lauroyl lactylate was applied topically to the EpiDerm^TM tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues. The mean relative tissue viability (% negative control) was reduced below 15% (11.9%) after 3 min treatment and below 15% (2.4%) after 60 min treatment. The positive and negative controls confirmed the validity of the study. Based on the results, the test item should therefore be classified as corrosive in accordance with CLP regulation 1272/2008 (Skin Corr 1A, H314).  Moreover, suitable in vivo test data is available showing that the target substance as a 10% concentrated solution did not induce any effects on the skin or eye of rabbits.

Based on the results, sodium lauroyl lactylate must be considered as corrosive to skin and eye is therefore classified as Skin Corr. 1A, H314; Eye Dam 1, H318 with a specific concentration limit of ≤ 10% to not induce any effects.

Justification for classification or non-classification

Based on the available data, the target substance needs to be classified as corrosive to skin and eye (Skin Corr. 1A, H314; Eye Dam. 1, H318) in accordance with CLP Regulation 1272/2008.