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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-11-14 to 2019-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

266-533-8

EC Name:

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

Cas Number:

66988-04-3

Molecular formula:

C24H44O6.Na

IUPAC Name:

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

Specific details on test material used for the study:

- Name: Sodium isostearoyl lactylate
- Chemical Name: Sodium isostearoyl-2-lactylate
- CAS No.: 66988-04-3
- Batch No.: 1827400007
- Molecular Weight: 450.59 g/mol
- Physical State at Room Temperature: clear viscous liquid
- Colour: light yellow
- Storage Conditions: room temperature
- Expiry Date: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Even though the test item is a liquid, it is very viscous and extremely difficult to pipet the exact amount required for this test (30 µL). Therefore, the test item was handled as a solid instead and 25 mg of the test item was used for this test. As the test item was very viscous, it was not possible to apply the test item directly on the tissue surface, so 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. 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 tissue surface. Next, the test item (25 mg; 39 mg/cm²) was 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. Despite the challenges of the application of the test item, the surface of the tested tissue samples could be entirely covered with the test item during the main experiment; therefore the outcomes of this study are not affected by the physical state of the test item.

In vitro test system

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

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

109.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

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	1.402	1.624	1.716
	1.445	1.546	1.420	0.294	0.260	0.225	1.406	1.666	1.760
OD570 (Blank Corrected)	1.400	1.488	1.379	0.244	0.210	0.181	1.362	1.584	1.675
	1.404	1.506	1.379	0.254	0.220	0.184	1.365	1.626	1.719
Mean OD570 of the Duplicates (Blank Corrected)	1.402	1.497	1.377	0.249	0.215	0.183	1.364	1.605	1.697
Total Mean OD570 of 3 Replicate Tissues (Blank Corrected)	1.425*			0.215			1.555
SD of Mean OD570of 3 Replicate Tissues (Blank Corrected)	0.063			0.033			0.172
Relative Tissue Viability [%]	98.4	105.0	96.6	17.4	15.1	12.8	95.6	112.5	119.0
Mean Relative Tissue Viability [%]	100.0			15.1**			109.1
SD of Relative Tissue Viability [%]***	4.4			2.3			12.1
CV of Relative Tissue Viability [%]	4.4			15.3			11.1

* 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_570nm of the negative control is 1.466, which fulfils the test acceptance criteria (mean absolute OD_570nm of 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	1.466	0.8 ≤ NC ≤ 2.8	pass
Mean Relative Viability [%] PC	15.1	≤ 20%	pass
SD of Relative Viability [%] (min-max)	2.3 -12.1	≤ 18%	pass

NC: Negative Control

PC: Positive Control

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the results obtained from this in vitro skin irritation study (OECD 439), the test item sodium isostearoyl lactylate can be considered to be non-irritant to the skin.

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 isostearoyl lactylate for 60 min followed by a 42-h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained and compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability of the test item (% negative control) was greater than 50% (109.1%). Based on this result, the test item sodium isostearoyl lactylate is considered to be non-irritating to the skin in accordance with UN GHS "No Category".