Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
other: In vitro skin irritation guideline study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-31 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD 439:In vitro skin irritation: Reconstructed Human Epidermis Test Method (adopted 22 July 2010)
Deviations:
yes
Remarks:
Listed below.
Qualifier:
according to
Guideline:
other: Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test ".
Deviations:
yes
Remarks:
Listed below.
Principles of method if other than guideline:
In vitro skin irritation test with Substance 3, NLP 500-090-6 using a human skin model.
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): Substance 3, NLP 500-090-6- Substance type: - Physical state: High viscous liquid- Composition of test material, percentage of components: - Approximately 57.5% bisGMA - Approximately 14.5% monomaleic adduct of bisGMA - Approximately 10% polymeric methacrylates (more Bisphenol-A’s + glycidyl in the chain)- Lot/batch No.:7008043- Expiration date of the lot/batch:01 June 2011- Stability under test conditions: Stable- Storage condition of test material: In refrigerator (2-8°C) protected from light-OTHER: Test substance preparationThe high viscous liquid test substance was slightly heated up to approximately 45°C before handling and cotton swabs were immersed in the test substance (see protocol deviation 1). The saturated swabs were stored refrigerated and protected from light upside down for approximately 2.5 hours until use. The saturated swabs were placed directly on top of the tissues and covered 80 to 90% of the skin tissue.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Skin Ethic
Justification for test system used:
As per OECD guideline.
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Saturated swabs with undiluted substance were placed into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μl PBS (negative control) and 3 tissues with 10 μl 5% SDS (positive control) respectively.
Duration of treatment / exposure:
The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. The substance could not be removed completely from the skin tissues. One of the tissues was damaged during removal of the substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37 °C.
Number of replicates:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls.

Test system

Details on study design:
Test systemEPISKIN Standard ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 11-EKIN-004. This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.RationaleIn the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).SourceSkinEthic Laboratories, Nice, France.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
> 50
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

In vivo

Irritant / corrosive response data:
The substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. The substance was stained yellow/brownish. The MTT solution was slightly discoloured, but did not turn blue / purple and no blue / purple precipitate was observed. Therefore it was concluded that the substance did not interact with MTT. The mean absorption at 570 nm measured after treatment with the substance and controls are presented in Table 1. The individual OD570 measurements are presented in Table 3. Table 2 shows the mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues was 94%. Since the mean relative tissue viability for the substance was above 50%, the substance is considered to be non-irritant. The positive control had a mean cell viability after 15 minutes exposure of 5%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1 Mean absorption in the in vitro skin irritation test with Modified Small Vinyl Ester

 

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean    

(OD570)

SD

Negative control

1.132

1.144

1.070

1.115                       ±   0.040

Test substance

1.111

*

0.980

1.046**                     ±   0.093

Positive control

0.036

0.052

0.091

0.060                       ±   0.028

 

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

 

* The tissue was damaged during removal of the test substance, and therefore the OD570 of this tissue is not reliable

** Based on two individually treated tissues

Table 2 Mean tissue viability in the in vitro skin irritation test with Modified Small Vinyl Ester

 

Mean tissue viability

(percentage of control)

 

Negative control

100

Modified Small Vinyl Ester

94**

Positive control

5

 

** Based on two individually treated tissues

Table 3 Individual OD measurements at 570 nm

 

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570 measurement 1

OD570 measurement 2

 

1.087

0.178

 

1.108

1.180

 

1.044

1.096

Modified Small Vinyl Ester

OD570 measurement 1

OD570 measurement 2

1.078

1.145

 

*

*

 

1.024

0.937

Positive control

OD570 measurement 1

OD570 measurement 2

 

0.035

0.037

 

0.042

0.063

 

0.093

0.089

 

OD = Optical density

Triplicate exposures are indicated by A, B and C.

* The tissue was damaged during removal of the test substance, and therefore the OD570of this tissue is not reliable

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
It is concluded that this test is valid and that the substance is non-irritant in the in vitro skin irritation test under the experimental conditions.
Executive summary:

The substance was a clear light yellow-greenish to light brown very high viscous liquid. The substance was applied on a saturated cotton swab, which was placed directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the substance. The relative mean tissue viability of two tissues obtained after 15 minutes treatment with the substance compared to the negative control tissues was 94%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be non-irritant. The positive control had a mean cell viability of 5% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly. Finally, it is concluded that this test is valid and that the substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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