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Diss Factsheets

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Jul - 08 Aug 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
April 13, 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 22, 2010
Deviations:
no
Principles of method if other than guideline:
In addition to the guidelines mentioned above, the study conduct followed the test strategy for determination of a corrosive property as given in the OECD Guideline for Testing of Chemicals No. 404, April 24, 2002 (“Acute Dermal Irritation/Corrosion”).

Test principle of the EpiDermTM Corrosivity-Test:
The EpiDermTM Corrosivity-Test is an in vitro test procedure used for the detection of the corrosive potential of a test substance.
Corrosive materials are identified by their ability to produce a decrease in cell viability, as determined, for example, by using the MTT reduction assay, below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.
The test material was applied unchanged topically to a three-dimensional human skin model comprising at least a reconstructed epidermis with a functional stratum corneum for 3 minutes and 1 hour. Negative and positive controls were added.
GLP compliance:
yes

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Plantapon LGC Sorb
- Lot/batch No.:CE02480006
- Physical state: liquid
- Analytical purity: 31.1%, aqueous sol.
- pH value (undiluted): ca. 6
- Stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:
other: not applicable
Strain:
other: not applicable
Details on test animals or test system and environmental conditions:
The test was conducted using the EpiDerm™ 200 Kit which consisted of 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm², cultured in Millicells® with a diameter of 1 cm.
The EpiDermTM model 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.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
single topical application of 50 µL for the corrosion test
single topical application of 30 µL for the irritation test
Duration of treatment / exposure:
Corrosion test: 3 minutes and one hour.
Irritation test: 1 hour followed by a 42-h post-exposure period.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
MESH COMPATIBILITY
Since the test item was a liquid, a nylon mesh was used as a spreading support. To exclude a reaction of the testitem with the mesh, the compatibility of the test item with the nylon mesh was first checked. For this purpose, the test item and the mesh were brought together on a slide and the reaction was observed after 60 minutes exposure, using a microscope. An interaction between test item and the mesh was not noticed. Therefore the test substance was applied using a mesh for spreading.

PRETEST [Direct MTT Reduction]
The decrease in cell viability as indicator for corrosivity was determined by using the MTT reduction assay (i.e. [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide].
Approximately 50 μL test item was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at 37 °C for 55 - 65 minutes. A negative control (50 μL of doubly distilled water) was tested concurrently. If the MTT solution colour or in case of water-insoluble test substances the border to the water-phase turns blue/purple, the test item is presumed to directly reduce MTT. The direct reduction of MTT by a test item interferes with the colour density produced by metabolic capacity of the tissue and would falsify the test results. In case that direct MTT reduction occurs one freeze-killed control tissue per exposure time is treated with, each, the test article and the negative control, in the same way as described below, additionally.

CORROSION TEST
On day of receipt EpiDermTM tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-item application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. Where applicable, one killed tissue per exposure time is treated to control for direct MTT reduction. Fifty microliter (50 μL) of undiluted test item were applied using a pipette. Control tissues were concurrently treated with 50 μL of doubly distilled water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC) or test substance (killed tissue control, KC). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of treatment and were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were again incubated for 3 hours. After incubation, the tissues were washed with PBS and the precipitated blue Formazan product produced by the tissues was extracted with Isopropanol over night at room temperature. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 3 microtiter wells filled with Isopropanol for each microtiter plate.

IRRITATION TEST
On day of receipt EpiDermTM tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced one hour later with fresh medium and preconditioning was continued for 18 +/- 3 hours. As for the corrosion assay, negative and positive controls (NC and PC) were added. Three tissues were treated with the test item, the PC and NC, respectively.
An amount of 30 μL of the undiluted liquid test item was applied using a pipette and a nylon mesh placed carefully onto the tissue surface afterwards.
Control tissues were concurrently applied with 30 μL of sterile PBS for negative control or with 30 μL of 5% SDS for positive control.
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 item 1 hour after start of application, blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. After rinsing, 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. Thereafter the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for further 18 ± 2 hours. The assay medium was then replaced by 0.3 mL MTT solution and the tissues were incubated again 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.


EVALUATION
The OD570 values determined for the various tissues are measures of their 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 corrosive or an irritating.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test is considered.
Assay acceptance criterion 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 the mean OD570 of the NC is ≥ 1.0; the meain OD570 however should not exceed 2.5.
Assay acceptance criterion for the positive control (PC):
Potassium hydroxide as 8.0 normal ready made solution is used as positive reference for the corrosion test. A 3-minute treatment with 8.0 n KOH usually reveals a mean relative tissue viability of about 20%. An assay is acceptable if mean relative tissue viability of the 3 min positive control is ≤ 30%. In the case of the irritation test where 5% SDS is used as PC, a viability of ≤ 20% is considered acceptable.
Assay acceptance criterion for tissue variability:
For every treatment, 2 or 3 tissues are treated in parallel for the corrosion and the irritation test, respectively. The intertissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is ≤ 0.3 for the corrosion test, and if the standard deviation of % of viability is ≤ 20 for the irritation test.
Assay acceptance criterion for killed controls (KC):
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35.

PREDICTION OF CORROSIVITY
A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.

Mean tissue viability (% negative control) => Interpretation
- after 3 minutes: < 50 <=> Corrosive
- after 3 minutes: ≥ 50 and after 1 hour: < 15 <=> Corrosive
- after 3 minutes: ≥ 50 and after 1 hour: ≥ 15 <=> Non-corrosive

PREDICTION OF IRRITATION
The irritant potential of the test item is predicted from the mean relative tissue viabilities compared to the concurrent NC tissues. A chemical is considered as "irritant", if the mean relative tissue viability with a test item is less than or equal to 50%.

Mean tissue viability (% negative control) => Interpretation
- ≤ 50 <=> Irritant
- > 50 <=> Non-irritant

HISTORICAL DATA
Historical ranges for negative controls, positive controls and viability were included in the study report.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: EpiDerm Skin Corrosivity Test
Basis:
other: mean viability of the test-item treated tissues after an exposure period of 3 minutes
Time point:
other: 3 minutes
Score:
104
Reversibility:
other: reversibility: not applicable
Remarks on result:
other: The mean viability of the test-item treated tissues determined after an exposure period of 3 minutes was 104%.
Irritation parameter:
other: EpiDerm Skin Corrosivity Test
Basis:
other: mean viability of the test-item treated tissues after an exposure period of 1 hour
Time point:
other: 1 h
Score:
105
Reversibility:
other: reversibility: not applicable
Remarks on result:
other: The mean viability of the test-item treated tissues determined after an exposure period of 1 hour was 105%.
Irritation parameter:
other: EpiDerm Skin Irritation Test
Basis:
other: mean viability of the test-item treated tissues after an exposure period of 1 hour and 42 hours post exposure
Time point:
other: 42 h
Score:
102
Reversibility:
other: reversibility: not applicable
Remarks on result:
other: The mean viability of the test-item treated tissues determined after an exposure period of 1 hour with about 42 hours post exposure was 102%.
Other effects:
Corrosivity test: For the positive control, mean viability after 3 minutes and 1 hour was 21% and 2%, respectively. These results confirmed the adequate performance of the test and the validity of the results.
Irritation test: For the positive control, mean viability was 14%. This result confirmed the adequate performance of the test and the validity of the results.

Any other information on results incl. tables

Test material

CORROSON TEST

Optical density (OD) after exposure for 3 minutes

OD570 [tissue 1]

OD570 [tissue 2]

Mean OD570

   

Viability [% of NC]

Negative control [NC]

2.109

2.079

2.094

 

100

Test Item

2.182

2.160

 2.171

 

104

Positive control [PC]

0.486

0.382

0.434

   

21

 

CORROSION TEST

Optical density (OD) after exposure for 1 hour

Negative control [NC]

2.113

2.154

2.134

   

100

Test Item

2.239

2.252

2.246

 

105

Positive control [PC]

0.047

0.038

0.042

   

2

Test material

IRRITATION TEST

Optical density (OD) after 1 hour ( + 42 h post treatment)

OD570 [tissue 1]

OD570 [tissue 2]

OD570 [tissue 3]

Mean OD570

 

Viability [% of NC]

Negative control [NC]

2.401

2.172

2.443

2.330

100

Test Item

2.602

2.225

2.313

2.380

102

Positive control [PC]

0.203

0.427

0.348

0.326

 

14

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU