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

Endpoint summary

Description of key information

In an in vivo skin irritation study (conducted in 1977), sodium isostearoyl lactylate was found to demonstrate skin irritating potential. However, in view of the exposure period (24 h), these results are not suitable for concluding on classification. Thus, an in vitro skin irritation study (in accordance with OECD Test Guideline 439) was subsequently performed, and the results showed that sodium isostearoyl lactylate can be considered as not irritating to the skin.

In an in vivo eye irritation study (also conducted in 1977), sodium isostearoyl lactylate was found to demonstrate mild eye irritating potential, but the reported effects in this study do not meet the GHS criteria for classification for eye irritation. A subsequent in vitro eye irritation study (in accordance with OECD Test Guideline 492) was performed, and the results showed that sodium isostearoyl lactylate can be considered as not irritating to the eye.

Taking the overall information from these in vitro and in vivo studies, it is concluded that sodium isostearoyl lactylate is non-irritating to the skin or the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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 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.

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:

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".

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

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".

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1977-08-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Study performed in accordance with the techniques specified in the US Regulations for the Enforcement of the Federal Hazardous Substances Act (Code of Federal Regulations, Title 16 Chapter II, 1976).

GLP compliance:

not specified

Specific details on test material used for the study:

- Test material name: Pationic ISL
- Source: C.J. Patterson Company
- Appearance: Thick clear yellow liquid with a slight odor
- Batch No. : Pl 06 24 77

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

N.A.

Type of coverage:

occlusive

Preparation of test site:

other: Intact and abraded

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL

Duration of treatment / exposure:

24 h

Observation period:

24 h and 72 h

Number of animals:

6 for intact and 6 for abraded skin experiments

Details on study design:

- For each sample, 0.5 mL of the undiluted test material (sodium isostearoyl lactylate; Pationic ISL) was applied under a one inch-square surgical gauze patch, two layers thick, to an intact skin area and an abraded ski area on each of six albino rabbits.
- The application sites were prepared by clipping the hair from the saddle area of the rabbits.
- After application of the patches, the trunk of each rabbit was wrapped with rubber dental damming which was secured with staples.
- An outer layer of gauze and tape was placed around the trunk of the animals. The animals were restrained in Newmann harnesses for 24 hours.
- At the end of the 24 h exposure period, the patches were removed and any residual sample was gently sponged from the skin with a moistened towel.
- The reactions were scored immediately after removal of the patches (24 h reading) and again two days later (72 h reading)

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: 24/72 h

Score:

7.17

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

72 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

72 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

72 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

72 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

72 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

72 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24 h

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

72 h

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24 h

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

72 h

Score:

2

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

72 h

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24 h

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #4

Time point:

72 h

Score:

2

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #5

Time point:

24 h

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #5

Time point:

72 h

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #6

Time point:

24 h

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #6

Time point:

72 h

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

animal #2

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

animal #3

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

animal #4

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

animal #5

Time point:

48 h

Remarks on result:

not measured/tested

Irritation parameter:

edema score

Basis:

animal #6

Time point:

48 h

Remarks on result:

not measured/tested

Irritant / corrosive response data:

- At the 24 h reading, all 12 sites were entirely blanched (whitened and thickened) with moderate peripheral erythema and moderate to severe oedema.
- By the 72 h reading, all 12 sites were coriaceous with well defined to moderate peripheral erythema and all sites exhibited slight to severe oedema.
- Fissuring was observed at one intact site at the 72 h observation period
- The Primary Irritation Index was found to be 7.17.

Table 1: Primary skin irritation scores in rabbits following a 24 h patch exposure to Pationic ISL

Skin	Time (h)	Score for Rabbit Number						Total Score	Average
		1	2	3	4	5	6
Erythema and Eschar Formation
Intact	24	4A	4A	4A	4A	4A	4A	24	4.00
	72	4E	4E	4E	4EC	4E	4E	24	4.00
Abraded	24	4A	4A	4A	4A	4A	4A	24	4.00
	72	4E	4E	4E	4E	4E	4E	24	4.00
Edema Formation
Intact	24	3	3	4	3	4	3	20	3.33
	72	3	2	3	2	3	3	16	2.67
Abraded	24	3	3	4	3	4	4	21	3.50
	72	3	3	3	3	4	3	19	3.17
Total									28.67

A: Entire site blanched (whitened and thickened) with moderate peripheral erythema

E: Entire site coriaceous with well-defined to moderate peripheral erythema

C: Fissuring

Interpretation of results:

GHS criteria not met

Conclusions:

Sodium isostearoyl lactylate is irritating in this study, based on a skin contact period far in excess of OECD test guideline instructions.

Executive summary:

In a skin irritation/corrosion study, 6 New Zealand white rabbits were exposed to sodium isostearoyl lactylate (Pationic ISL) on intact skin for 24 hours. The test animals were then observed for 24 and 72 h following treatment. The rabbits experienced blanching (whitening and thickening), fissuring and edema. The Primary Irritation Index was found to be 7.17. With a view to the exposure period, these results are not suitable for concluding on classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1977-08-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Principles of method if other than guideline:

The study was conducted in accordance with the techniques specified in the Regulations for the Enforcement of the Federal Hazardous Substances Act (Code of Federal Regulations, Title 16 Chapter II, 1976).

GLP compliance:

no

Specific details on test material used for the study:

- Test material name: Pationic ISL
- Source: C.J. Patterson Company
- Appearance: Thick clear yellow liquid with a slight odor
- Batch No. : Pl 06 24 77

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL

Duration of treatment / exposure:

Up to 72 h

Observation period (in vivo):

24, 48 and 72 h

Number of animals or in vitro replicates:

6

Details on study design:

- For each sample, 0.1 mL of the undiluted test material was applied to the right eye of each of six New Zealand White rabbits from Sweetwater Farms. The opposite eyes were untreated and served as controls.
- Examinations for gross signs of eye irritation were made at 24, 48 and 72 h following application.
- Scoring of irritation effects was according to the method of Draize, in which corneal, iris and conjunctival effects are scored seperately.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.6

Max. score:

3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.6

Max. score:

3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.6

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.6

Max. score:

3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2.6

Max. score:

3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1.6

Max. score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.6

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1.6

Max. score:

3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

- Irritative effects within 24 h following application included mild corneal opacity in 5 rabbits, mild iritis in three rabbits and mild to moderate conjunctivitis in all rabbits.
- By termination of the study, mild corneal opacity was noted in three rabbits, mild iritis was noted in three rabbits and mild to moderate conjunctivitis was observed in all six rabbits.

Table 1: Eye Irritation scores in albino rabbits following application of 0.1 mL of Pationic ISL.

Rabbit Number	Time (h)	Cornea		Iris	Conjunctivae			Total Score*
		Opacity	Area		Erythema	Swelling	Discharge
1	24	1	2	1	2	2	2	27
	48	1	1	1	3	1	2	22
	72	1	1	1	2	1	2	20
2	24	0	0	0	2	1	2	10
	48	0	0	0	2	1	1	8
	72	0	0	0	1	0	0	2
3	24	1	3	1	2	2	3	34
	48	1	2	1	2	1	3	27
	72	1	1	1	1	0	1	14
4	24	1	2	1	2	2	3	29
	48	1	2	1	3	1	1	25
	72	1	1	1	2	1	1	18
5	24	1	1	0	1	1	1	11
	48	0	0	0	1	0	0	2
	72	0	0	0	0	0	0	0
6	24	1	2	0	2	2	1	20
	48	0	0	0	2	1	1	8
	72	0	0	0	1	0	0	2

* Total score is the sum of the following 3 sub-totals: (a) degree of opacity x area involved x 5, (b) iris score x 5 and (c) sum of scores for erythema, swelling and discharge x 2

Total possible score = 110.

Interpretation of results:

GHS criteria not met

Conclusions:

An in vivo eye irritation rabbit study of sodium isostearoyl lactylate showed mild eye irritant effects starting at 24 h after application of the substance. There was no increase in severity of the effects over the 72-h observation period, and the degree of corneal opacity and of conjunctival effects was reduced by 72 h in a number of rabbits. Based on these results and the GHS classification criteria for eye irritation, sodium isostearoyl lactylate does not warrant classification for eye irritation.

Executive summary:

In an in vivo eye irritation study, 6 New Zealand white rabbits were exposed to 0.1 mL of the test substance sodium isostearoyl lactylate (undiluted) and were observed 24, 48 and 72 h following application of the test substance. Mild irritative effects such as corneal opacity, iritis and conjunctivitis were observed in several rabbits within 24 h following application. Three out of six tested rabbits (i.e. 50%) exhibited corneal opacity as well as iritis with respective mean scores of 1 following grading at 24, 48 and 72 h after application. As for conjunctival redness (as reported as erythema in the study), two out of six tested rabbits (i.e. 33%) exhibited mean scores of 2 or higher, and no tested rabbits exhibited conjunctival oedema (as reported as swelling in the study) with a mean score of 2 or higher. There was no increase in severity of the effects over the 72-h observation period, and the degree of corneal opacity and of conjunctival effects was reduced by 72 h in a number of rabbits. Based on these results and the GHS classification criteria for eye irritation, sodium isostearoyl lactylate does not warrant classification for eye irritation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-07 to 2018-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

29 June 2015

Deviations:

yes

Remarks:

The deviation did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

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.: Pationic ISL (Lot 1731200025)
Molecular weight: 450.59 g/mol
Aggregate State at room temperature: clear viscous liquid
Colour: light yellow
Storage Conditions: room temperature
Expiry Date: unopened container may be used for up to 12 months

Species:

human

Details on test animals or tissues and environmental conditions:

TEST TISSUE DETAILS
- Three-dimensional RhCE EpiOcular™ (MatTek): normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical, and physiological properties of the human corneal epithelium.
- The test item was pre-incubated at 37 ± 2 °C for 15 ± 1 min before deciding which treatment protocol (for liquid or solid) was to be used. The liquid test material was very sticky, so a nylon mesh was used as a spreading aid to ensure even distribution of the test material .Mesh compatibility was tested by applying 50 uL of the test item to a nylon mesh placed on a slide. After 60 minutes, the mesh was examined microscopically and no interaction between the nylong mesh and the test material was found.

Vehicle:

not specified

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 uL aqua dest.
- Lot/batch no. (if required): Lot RNBG3519, Sigma
- Purity: not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 uL methyl acetate
- Lot/batch no. (if required): Lot S6943111, CAS No. 79-20-9, Merck
- Purity: not reported

Duration of treatment / exposure:

30 ± 2 mins at 37 ± 2 °C, 5.0% CO2 / 95% air

Duration of post- treatment incubation (in vitro):

12 ± 2 min at room temperature post-rinse after exposure period; 120 ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% air post-treatment plate

Number of animals or in vitro replicates:

2 replicates per dose

Details on study design:

- Details of the test procedure used
Upon receipt of the EpiOcular™, the tissues (provided in inserts) were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air. Then the tissues were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air for 16 - 24 h. After the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye. Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. The test item was applied on the tissue inserts placed on a sterile surface. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one minute intervals. After dosing, the tissues were placed back into the culture medium. Then the 6-well plate(s) were incubated for 30 ± 2 min at 37 ± 2 °C, 5.0% CO2 / 95% air. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS and this step was also done sequentially, following the same order as the dosing of the tissue. Excess DPBS was removed by decanting the tissue and blotting bottom with blotting paper. After rinsing, the tissues were transferred to and immersed in a pre-prepared 12-well “postsoak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 12 ± 2 min at room temperature. Afterwards, the tissues were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The tissues were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 120 ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% air. After this incubation period excess medium was removed by blotting bottom on absorbent paper before the tissues were transferred in a pre-prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% air. After the 3 h MTT incubation period the tissues were removed, the bottom of the tissues blotted on blotting paper and then transferred into new 24-well “extraction plates“ containing 2 mL of isopropanol. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were pierced and the liquid within each tissue was decanted into the well from which it was taken. Then the tissues were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged (400g, 5min, room temperature) to eliminate the fragments and avoid further possible interference with the absorbance readings. For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.
- RhCE tissue construct used, including batch number :
-The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
-1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 27023)
-1x bottle EpiOcularTM assay medium (Lot No.: 021218ISA)
-1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 092817MGKA)
- Doses of test chemical and control substances used :
- Test chemical: 50 uL Sodium isostearoyl lactylate
- Positive control: 50 uL methyl acetate
- Negative control: Aqua dest.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) :
- Exposure: 30 ± 2 mins
- Post-exposure immersion: 12 ± 2 mins
- Post-exposure incubation: 120 ± 15 min
- Description of any modifications to the test procedure : The test item was a very sticky liquid so a nylon mesh was used for application of the test item. The protocol for liquid test items instead of solids was used for the experimental procedure.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : all performed in duplicate
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570 using a filter band pass of maximum ± 30 nm in a spectrophotometer (isopropanol used as a blank)
- Description of the method used to quantify MTT formazan : During the pre-experimental phase, to check the non-specific MTT-reducing capability of the test item 50 µL of the test item were mixed with 1 mL MTT medium and incubated for 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2 / 95% air. If the mixture turned blue/purple, the test item was presumed to have reduced MTT. The part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues if the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value. In case the mean relative tissue viability of the test item treated tissues (TM) is below 60%, NSMTT correction would not be required as it would not affect the final prediction of the study. For quantitative correction of results, two killed tissues were treated with 50 µL of the test item (KT) and one tissue was treated with 50 µL of the negative control (Aqua dest.; KU), respectively.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : The mean OD of the two negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability was calculated according to the following formula: Relative viability (%) = [mean ODtest item / positive control / mean ODnegative control] * 100. For the test item and the positive control the mean relative viability ± relative standard deviation of the two individual tissues was calculated and used for classification as described in the prediction model below. Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the concurrent negative control tissues treated with Aqua dest. The test item is considered to be irritant to the eye if the relative tissue viability is less or equal to 60%, but it cannot be differentiated between UN GHS [7] “Category 1” or “Category 2”. The test item is considered to be non-irritant in accordance with UN GHS “No Category” if relative tissue viability is higher than 60%. See Table 1 in "Any other information on materials and methods incl. tables".
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : yes, generated 2012-2016
- Complete supporting information for the specific RhCE tissue construct used : The MatTek EpiOcular™ model has been widely used as a research and testing model for many years
- Reference to historical data of the RhCE tissue construct : none
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : The MatTek EpiOcular™ model has been widely used as a research and testing model for many years
- Positive and negative control means and acceptance ranges based on historical data : not specified
- Acceptable variability between tissue replicates for positive and negative controls : mean absolute OD570 nm of the negative control is > 0.8 and < 2.5; mean relative tissue viability of the positive control is < 50%
- Acceptable variability between tissue replicates for the test chemical: relative tissue viability difference of replicate tissues is < 20%

Irritation parameter:

in vitro irritation score

Remarks:

Mean tissue viability (% of negative control) non-specific colour of additional viable tissues-living corrected

Run / experiment:

Mean of 2 test item runs

Value:

71.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (2.011).
- Acceptance criteria met for positive control: Yes - The mean relative tissue viability (% negative control) of the positive control was < 50% (31.5%)

Table 2. Result of the Test Item Sodium Isostearoyl Lactylate

Name	Negative Control		Positive Control		Test item
Tissue	1	2	1	2	1	2
OD570 values	2.037	1.988	0.605	0.715	1.567	1.342
	2.021	1.998	0.599	0.732	1.594	1.337
OD570 values (blank-corrected)	1.993	1.945	0.562	0.972	1.523	1.299
	1.978	1.955	0.556	0.689	1.551	1.294
Mean of the duplicates	1.986	1.950	0.559	0.680	1.537	1.297
Mean OD	1.968*		0.620		1.417
TOD-TT	-		-		1.413
SD of the mean OD	0.025		0.086		0.170
Tissue viability (%)	100.9	99.1	28.4	34.6	78.1	65.9
Mean tissue viability (%)	100.0		31.5**		72.0
Relative tissue viability difference (%)***	1.8		6.2		12.2
Mean tissue viability (%) - NSC-living corrected	-		-		71.8

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

**Mean relative tissue viability of the positive control tha tis considered acceptable for this study is < 50%

***Relative tissue viability difference of replicate tissues that is considered acceptable for this study is < 20%

Table 3. Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NK	2.011	0.8 < NK < 2.5	pass
Mean Relative Viability PC (%)	31.5	< 50%	pass
Max. Difference of % Viability (%)	12.2	< 20%	pass

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item Sodium isostearoyl lactylate showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category” for eye irritation

Executive summary:

A test using the three-dimensional RhCE EpiOcular™ (MatTek) consisting of normal, human derived epidermal keratinocytes and mimicking the histological, morphological, biochemical and physiological properties of the human corneal epithelium was conducted to evaluate the eye hazard potential of a test chemical. This evaluation is based on the test chemical's ability to induce cytotoxicity in a reconstructed human cornea-like epithelium (RhCE) tissue construct after topical application, expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT. In this study under the given conditions the test item Sodium isostearoyl lactylate showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category” for eye irritation.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Principles of method if other than guideline:

- Principle of test: Use immortalized human corneal epithelial (HCE) cells cultured onto a polycarbonate membrane at the air liquid interface and forming a multi-layered epithelium in order to evaluate the in vitro eye irritancy potential of a substance.
- Short description of test conditions: HCE epithelia treated topically with 30 ± 2 µL of liquid substance for 1 hour at room temperature. Post-treatment, HCE tissues were rinsed with sterile PBS and incubated with new maintenance medium for 16 hours at 37 °C, 5 % CO2. 3 tissues used for each treatment. Absolute ethanol served as the positive control and PBS as the negative control.
- Parameters analysed / observed: Tissue viability assessed using MTT reduction assay reagent.

GLP compliance:

not specified

Specific details on test material used for the study:

OTHER SPECIFICS: Sodium isostearoyl lactylate and all other test substances came from L'Oreal chemicals library
Applied at 15% dilution.

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability : The test substance was a liquid.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The SkinEthic™ HCE model was purchased from SkinEthic Laboratories
(45, Rue St Philippe, 06000 Nice, France). The model was
composed of immortalized human corneal epithelial cells cultured
in a chemically defined medium and seeded on a synthetic membrane
at the air–liquid interface. The tissue structure obtained
was a multilayered epithelium resembling the in vivo epithelium
(Fig. 1a) representing about 5–7 cell layers and a surface area of
0.5 cm2
. As in vivo, columnar cells and wing cells were present
(Fig. 1d). The epithelial tissues were characterized by the presence
of specific ultra structural figures such as intermediate filaments,
mature hemi-desmosomes and desmosomes (Fig. 1b, c, e, and f).
Specific cytokeratins 64kD (K.3) were also described (Nguyen
et al., 2003). Tissues were shipped in agarose semi-solid culture
medium. Upon arrival they were transferred to a new maintenance
medium, 1 mL/well in 6 well plates and incubated at 37 C, 5% CO2
in a humidified incubator. The medium was renewed 24 h later.

Vehicle:

not specified

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 ± 2 uL (representing 60 uL/cm2)

Duration of treatment / exposure:

1 hour

Duration of post- treatment incubation (in vitro):

16 hours

Number of animals or in vitro replicates:

3 tissues used for each treatment

Details on study design:

- Details of the test procedure used : HCE epithelia were topically treated with 30 ± 2 lL (representing 60 lL/cm2) for liquid pipetable substances. The tissues were treated for 1 h at room temperature (24 well plates with 300 lL/well of maintenance medium). After treatment, HCE tissues were rinsed with sterile D-PBS (3 times 5 mL using a multistep pipette, 500 lL per delivery) and incubated with new maintenance medium (300 lL/well) for an additional 16-h period at 37 °C, 5% CO2 in a humidified incubator. Three independent runs per substance were used. Three tissues were used for each treatment for all sets. Triton X-100 0.2% in water was used as the batch control (BC), absolute ethanol was used as the positive control (PC) and PBS as the negative control (NC).
- RhCE tissue construct used, including batch number : The SkinEthic™ HCE model was purchased from SkinEthic Laboratories (45, Rue St Philippe, 06000 Nice, France). The model was composed of immortalized human corneal epithelial cells cultured in a chemically defined medium and seeded on a synthetic membrane at the air–liquid interface. The tissue structure obtained was a multilayered epithelium resembling the in vivo epithelium representing about 5-7 cell layers and a surface area of 0.5 cm2. No batch number reported.
- Doses of test chemical and control substances used : 30 ± 2 uL, representing 60 uL/cm2; doses of control substances not reported.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : exposure - 1 hour at room temperature; post-exposure incubation period - 16-h period at 37 °C, 5% CO2 in a humidified incubator
- Justification for the use of a different negative control than ultrapure H2O (if applicable) : none given
- Justification for the use of a different positive control than neat methyl acetate (if applicable) : none given
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : To detect a possible interaction, the substances
(30 lL or 30 mg) were incubated with 0.5 mg/mL MTT in culture medium (300 lL 24-well plates). The appearance of a blue/purple color after 3 h incubation, indicate a positive interaction. If the test substance has a potential to directly reduce MTT, the assay was performed in parallel on viable and on killed tissues in order to evaluate the non-specific OD and correct final results. Dead epithelia were obtained by pre-incubation of the tissues with distilled water (1 mL/well in 6-well plates). The subsequent use of water-killed tissues followed the same protocol steps than living tissues. Colored substances could be visualized directly and after 10% dilution (w/w) in water. At this stage, simple unaided eye detection was sufficient. For coloring substances, additional control tissues were treated in accordance with all of the protocol steps excluding MTT incubation (replaced by 3 h incubation in maintenance medium without MTT). These controls enabled the final quantification of non-specific absorbance due to the remaining color in/on the tissue after rinsing.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : Three tissue replicates per test chemical and controls
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570 nm (filter band pass ± 30 nm)
- Description of the method used to quantify MTT formazan : Tissues were quickly blotted on absorbent paper and incubated with 0.5 mg/mL MTT in maintenance medium (24-well plates, 300 lL/well) for 3 h at 37 °C, 5% CO2 in a humidified atmosphere. Tissues were then transferred to 1.5 mL of isopropanol (750 lL per well and 750 lL added onto each tissue) for 2 h at room temperature and protected from evaporation and light (formazan extraction). 200 lL aliquots of formazan extracts were transferred to flat bottom 96-well plates for optical density measurement at 570 nm (filter band pass ± 30 nm) using a spectrophotometer micro-plate reader and isopropanol as the blank.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : According to historical values, NC met the acceptance criteria if the optical density values were P0.7 with readings recorded at 570 nm ban pass ± 30 nm. BC data met the acceptance criteria if the mean viability, expressed as a % relative to the NC, was 640%. PC data met the acceptance criteria if the mean viability, expressed as a % relative to the NC, was 610%. Based on the viability (expressed as %) endpoint, a cut-off was set to a 50% reduction in viability in relation to the NC. If the % viability was >50%, the substance was predicted as Non-Irritant (EU classification: no label; GHS classification: No Category). If the % viability 650%, the substance was predicted as Irritant (EU classification: R41 or R36; GHS classification: Category 1 or Category 2).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : yes
- Complete supporting information for the specific RhCE tissue construct used : not specified
- Reference to historical data of the RhCE tissue construct : not specified
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : not specified
- Positive and negative control means and acceptance ranges based on historical data : yes
- Acceptable variability between tissue replicates for positive and negative controls : yes
- Acceptable variability between tissue replicates for the test chemical: yes

Irritation parameter:

in vitro irritation score

Remarks:

mean % viability

Run / experiment:

Based on 3 runs

Value:

82.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

(15% concentration)

Other effects / acceptance of results:

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

Interpretation of results:

GHS criteria not met

Conclusions:

Long exposure time assay adapted to the SkinEthic(TM) HCE model visualized with MTT-formazan was demonstrated on 102 substances from L'Oreal chemical library, which included test substance sodium isostearoyl lactylate (tested at 15% concentration; diluted in water), to assess eye irritation. Sodium isostearoyl lactylate did not demonstrate eye irritation.

Executive summary:

Long exposure time assay adapted to the SkinEthic(TM) HCE model visualized with MTT-formazan was demonstrated on 102 substances from L'Oreal chemical library, which included test substance sodium isostearoyl lactylate (tested at 15% concentration), to assess eye irritation. Sodium isostearoyl lactylate did not demonstrate eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are several in vitro and in vivo studies examining the potential of sodium isostearoyl lactylate to induce skin irritation and eye irritation. In an in vivo skin irritation/corrosion study (Hilltop Research, 1977), 6 New Zealand white rabbits were exposed to sodium isostearoyl lactylate on intact skin for 24 hours. The test animals were then observed for 24 and 72 h following treatment. The rabbits experienced blanching (whitening and thickening), fissuring and oedema. The Primary Irritation Index was found to be 7.17. In view of the exposure period (24 hours), these results are not suitable for concluding on classification. Thus, an in vitro skin irritation study was subsequently performed in accordance with OECD Testing Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). The mean tissue viability was greater than 50% (109.1 %). Based on these results, sodium isostearoyl lactylate can be considered as non-irritating to the skin.

In an in vivo eye irritation study (Hilltop Research, 1977), 6 New Zealand white rabbits were exposed to 0.1 mL of the test substance sodium isostearoyl lactylate (undiluted) and were observed 24, 48 and 72 h following application of the test substance. Mild irritative effects such as corneal opacity, iritis and conjunctivitis were observed in several rabbits within 24 h following application. Three out of six tested rabbits (i.e. 50 %) exhibited corneal opacity as well as iritis with respective mean scores of 1 following grading at 24, 48 and 72 h after application. As for conjunctival redness (as reported as erythema in the study), two out of six tested rabbits (i.e. 33 %) exhibited mean scores of 2 or higher, and no tested rabbits exhibited conjunctival oedema (as reported as swelling in the study) with a mean score of 2 or higher. There was no increase in severity of the effects over the 72-h observation period, and the degree of corneal opacity and of conjunctival effects was reduced by 72 h in a number of rabbits. These reported effects are considered as insufficient to meet the GHS criteria for classification of eye irritation. A subsequent in vitro test for eye irritation performed in accordance to OECD test guideline 492 revealed no eye irritation potential of sodium isostearoyl lactylate. Based on these results, sodium isostearoyl lactylate can be considered as non-irritating to the eye.

Justification for classification or non-classification

In vivo data showed skin irritative effects of sodium isostearoyl lactylate, but the reported effects are not suitable for classification for skin irritation under GHS criteria (due to the extended 24-hour exposure period). The in vitro test on skin irritation performed in accordance to OECD Test Guideline 439 revealed no skin irritation potential of sodium isostearoyl lactylate. Based on these results, sodium isostearoyl lactylate can be considered as non-irritant to the skin and does not warrant classification for skin irritation.

In vivo data showed mild eye irritative effects of sodium isostearoyl lactylate, but they do not meet the GHS criteria for classification for eye irritation. An in vitro test for eye irritation performed in accordance to OECD Test Guideline 492 revealed no eye irritation potential of sodium isostearoyl lactylate. Based on these results, sodium isostearoyl lactylate can be considered as non-irritating to the eye and does not warrant classification for eye irritation.