Oligomerisation products of sucrose with ethylene oxide and methyloxirane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Feb 2011 - 02 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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 for Testing of Chemicals No. 439, July 22, 2010 (“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:

Oligomerisation products of sucrose with ethylene oxide and methyloxirane

EC Number:

701-303-7

Molecular formula:

C12 H14O11. ( C3 H6 O )(b+d+f+h+j+l+n+p).(C2 H4 O)(a+c+e+g+i+k+m+o) ; 1 < a+c+e+g+i+k+m+o < 16.5; 1 < b+d+f+h+j+l+n+p < 16.5

IUPAC Name:

Oligomerisation products of sucrose with ethylene oxide and methyloxirane

Details on test material:

- Name of test material (as cited in study report): Sucrose, ethoxylated and propoxylated (>1 < 16.5 mol EO and >1 < 16.5 mol PO)
-Test item number: 11/0043-1
- Physical state: Liquid, viscous / colorless, clear
- Analytical purity: >99%
- Lot/batch No.: T35/035/10
- Stability under test conditions: guaranteed by the sponsor
- Storage condition of test material: room temperature, protect against humidity
- pH-value of the undiluted test substance: 5

Test animals

Species:

other: not applicable; in vitro test

Strain:

other: not applicable; in vitro test

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- The test system (target tissue): three dimensional human epidermis model.
The EpiDerm™ model (Epi-200) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized 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 EpiDem™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
- Supplier: MatTek Corp., Ashland MA, USA.

Test system

Type of coverage:

open

Preparation of test site:

other: in vitro test (direct application)

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro test; 30 µL PBS, sterile was used as negative control (NC)

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µl
- Concentration: undiluted test substance

Duration of treatment / exposure:

1 hour (25 min at room temperature/35 min at 37°C)

Observation period:

42±4 hours

Number of animals:

3 tissues were used per treatment for the test substance, the negative control (NC) and the positive control (PC)

Details on study design:

Test procedure:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. 30 μL of the undiluted test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

DATA EVALUATION:
-Principle: the OD570 value determined for each tissue was taken as a measure of its viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is irritant.
-Calculation of individual and mean optical densities: the individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.
-Tissue viability: the quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent

ACCEPTANCE CRITERIA:
-Assay acceptance criteria for the negative control (NC): the absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if (1) the mean OD570 of the NC is ≥ 1.0 and (2) the mean OD570 of the NC ≤ 2.5.
- Assay acceptance criterion for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Assay acceptance criterion for tissue variability: for every treatment, 3 tissues are treated in parallel. The intertissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.

EVALUATION OF RESULTS:
Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

HISTORICAL CONTROL DATA
Historical control values of negative and positive controls, gathered over an appropriate time period, were available. These data demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria for the test system.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

106

In vivo

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria, the test substance does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.

Any other information on results incl. tables

Table 1: Findings of the skin irritation test.

Test substance		tissue 1      tissue 2      tissue 3	mean             SD
NC	mean OD570	1.939           1.755           1.975	1,890
	viability [% of NC]	102.6            92.9            104.5	100             6.24
11/0043-1	mean OD570	1.990           1.997           2.030	2,006
	viability [% of NC]	105.3           105.7           107.4	106             1.12
PC	mean OD570	0.111           0.124           0.133	0,123
	viability [% of NC]	5.9               6.6               7.1	7                0.59

 

Table 2: Historical control values of the negative control (NC), the positive control (PC), and the viability. (SD= standard deviation)

Historical range of NC (OD570); Historical period: Mar 2009 - Apr 2011
Mean OD	SD	Mean+2SD	Mean-2SD
1.933	0.202	2.34	1.53
Historical range of PC (OD570); Historical period: Mar 2009 - Apr 2011
Mean OD	SD	Mean+2SD	Mean-2SD
0.120	0.022	0.16	0.08
Viability (%); Historical period: Mar 2009 - Apr 2011
Mean %	SD	Mean+2SD	Mean-2SD
6.3	1.22	8.69	3.83

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS