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Toxicological information

Skin irritation / corrosion

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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:
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,2`-Oxybisethanol, ethoxylated and propoxylated (>1< 4.5 mol EO and >1< 4.5 mol PO)
- Physical state: liquid, colorless, clear
- Analytical purity: >99%
- Test item number: 11/0042-1
- Lot/batch No.: T35/034/10
- Storage condition of test material: room temperature
- pH-value of the undiluted test substance: 6

Test animals

Species:
other: not applicable; in vitro test
Strain:
other: not applicable; in vitro test
Details on test animals 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. Thirty microliter (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 in the incubator 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 used 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 is ≤ 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

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
first run
Value:
83
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
second run
Value:
113

In vivo

Irritant / corrosive response data:
In the first run the SD of %-viability of the test substance was out of the acceptance range. Therefore a second run was performed where all acceptance criteria were met. Based on the observed results and applying the evaluation criteria, it was concluded, that 2,2'-Oxybisethanol, ethoxylated and propoxylated (>1 < 4.5 mol EO and >1 <4.5 mol PO) does not show a skin irritation potential in the EpiDermTM skin irritation test under the test conditions chosen.

Any other information on results incl. tables

Table 1: Findings in the first run 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

11/0042-1

 mean OD570

1.857           1.830           1.030

2.006

viability
[% of NC]

98.2             96.8             54.5

83              24.87

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: Findings in the second run of the skin irritation test.

Test
substance

 

tissue 1      tissue 2      tissue 3

mean             SD

NC

mean OD570

1.620           1.810           1.927

1.786

viability
[% of NC]

90.7             101.4          107.9

100             8.68

11/0042-1

 mean OD570

1.984           2.055          2.006

2.015

viability
[% of NC]

111.1           115.1          112.4

113             2.03

PC

mean OD570

0.100           0.082          0.089

0.090

viability
[% of NC]

5.6               4.6               5.0

5                 0.51

 

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

Historical range of 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