Sucrose and glycerol, reaction products with C12-18, C18unsatd. fatty acids

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-21 to 2011-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sucrose and glycerol, reaction products with C12-18, C18unsatd. fatty acids

Molecular formula:

Representative, generic structures are given in "structural formula" wherein R = H or fatty acid residue and R' = sucrose residue, glycerol residue, H or alkali. Additional citric acid resp. its salt is present.

IUPAC Name:

Sucrose and glycerol, reaction products with C12-18, C18unsatd. fatty acids

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Test animals

Species:

other: Human skin model: EpiSkin™

Strain:

other: EpiSkin™ Kit Lot No.: 11-EKIN-034

Test system

Type of coverage:

other: not applicable in this in vitro test system

Preparation of test site:

other: not applicable in this in vitro test system

Vehicle:

water

Controls:

other: refer to "details on study design"

Amount / concentration applied:

TEST MATERIAL
- Amount applied to each EpiSkin™ insert of the treatment group: 10 mg of test substance

VEHICLE
- Amount applied to each EpiSkin™ insert of the treamtent group: 15 μL of deionised water

Duration of treatment / exposure:

15 ± 1 min

Observation period:

refer to "details on study design"

Number of animals:

refer to "details on study design"

Details on study design:

TEST MODEL
For the test an EpiSkin™ Reconstituted Human Epidermis model was used. The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues are cultured on specially prepared cell culture inserts.

PREPARATION OF TEST SYSTEM
EpiSkin™ tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate, they were delivered one day prior to the experiment, transferred to 12-well plates with maintenance medium and prewarmed for 23 hours.

TREATMENT
Three sets of triplicate EpiSkin™ tissues contained in 12-well plates were treated respectively with the negative and positive controls (10 μL were dosed per tissue) and with test substance. Due to its waxy consistency, firstly 10 mg of test substance were weighed on top of the stamp of a syringe, wetted with 15 μL of deionised water, and then evenly applied to each of triplicate tissues used for treatment. Incubation took place for 15 ± 1 min at 37 ± 1.5 °C and 5 ± 0.5 % CO2.
At the end of the treatment period the tissue inserts were immediately removed from the 12-well plate and gently rinsed with PBS to remove any
residual test substance. The rinsed inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C and 5 ± 0.5 % CO2.

MTT ASSAY FOR CELL VIABILITY
After 42 hours of incubation the tissues were transferred for the MTT cell viability assay to 12-well plates for a 3 hour incubation period (37 ± 1.5 °C and 5 ± 0.5 % CO2). Then, and following removal of MTT and rinsing, the tissue sampels were immersed into vials containing 0.5 mL extractant solution (isopropanol / 2 N HCl 49:1 (v/v)). Then the tissue samples were completely covered by isopropanol, sealed to inhibit isopropanol evaporation and the formazan salt was extracted for approximately 69 hours in the refrigerator.
Per each tissue sample 2 x 200 μL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.

CONTROL OF MTT REDUCING ACTIVITY OF TEST ITEM
MTT reducing capability of the test substance was tested: ~25 mg of the test item were added to 1 mL of MTT-containing medium and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: tissue viability: MTT reduction measured as Formazan salt formation

Remarks on result:

other:

Remarks:

Basis: other: mean of all replicates. Time point: 15 min. Reversibility: other: not applicable. Remarks: relative absorbance at 570 nm for test substance: 87.5 % (threshold for irritancy: ≤ 50 %),. (migrated information)

In vivo

Irritant / corrosive response data:

Treatment of three EpiSkin™ tissue inserts with the test substance resulted in a mean relative absorbance value of the test substance at 570 nm, corresponding to the cell viability, of 87.5 % as compared to 100.0 % mean relative absorbance for the negative control and 33.0 % for the positive control. The threshold value for irritancy is ≤ 50 %, consequently the test substance was non irritant to skin in this test system.

Any other information on results incl. tables

Results after treatment with Sucroglyceride C12-18, C18unsatd. and controls

Dose group	Treatment Interval	Absorbance 570 nm Tissue 1	Absorbance 570 nm Tissue 2	Absorbance 570 nm Tissue 3	Mean Absorbance of 3Tissues	Relative Absorbance [%] Tissue 1, 2 + 3**	Standard Deviation[%]	Rel. Absorbance [% of Negative Control]***
Negative Control	15 min	0.801	0.814	0.801	0.805	99.5 101.1 99.5	0.9	100.0
Positive Control	15 min	0.269	0.273	0.255	0.266	33.4 33.8 31.7	1.1	33.0
Test Substance	15 min	0.697	0.698	0.719	0.705	86.6 86.6 89.3	1.6	87.5

* Mean of two replicate wells after blank correction

 

 ** relative absorbance per tissue [rounded values]:

 

100 x (absorbance_tissue)

__________________________________________________________

 (mean absorbance_{negative control})

 

 *** relative absorbance per treatment group [rounded values]:

 

100 x (mean absorbance_{test substance})

___________________________________________________________

 (mean absorbance_{negative control})

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Treatment of RHE (Reconstituted Human Epidermis) with Sucroglyceride C12-18, C18unsatd. resulted in a mean relative absorbance of 87.5% as compared to 100.0% mean relative absorbance for the negative control. According to the evaluation scheme used in the test system, a substance is considered irritant at a mean realtive absorbance ≤ 50%. Thus, the test substance Sucroglyceride C12-18, C18unsatd. was graded as not irritating to the skin in this in vitro assay.

Executive summary:

In an in vitro Human Skin Model Test, performed according to the OECD Test Guideline 439, the potential to cause irritation upon first contact with the skin was assessed by application of the test substance to RHE (Reconstituted Human Epidermis). 10 mg of Sucroglyceride C12 -18, C18unsatd. were applied together with 15 μL of water to an area of approximately 0.38 cm² of RHE and incubated for 15 min.

For evaluation of the potential of the test substance to cause skin irritation, the samples of treated reconstituted skin were then exposed to MTT for 3 h. The potential to reduce MTT and form a blue Formazan salt was assessed by extraction of Formazan over 69 h and subsequent determination of the absorbance at 570 nm.

Treatment with a 5% solution of SLS (Sodium lauryl sulphate) in deionised water, used as positive control, resulted in a mean relative absorbance of 33.0%, thus showing the normal functionality of the test system.

Treatment with the test substance resulted in a mean relative absorbance of 87.5% as compared to 100.0% mean relative absorbance for the negative control. According to the evaluation scheme used in the test system, a substance is considered irritant at a mean realtive absorbance ≤ 50 %. Thus, the test item Sucroglyceride C12 -18, C18unsatd. was graded as not irritating to the skin in this in vitro assay.