Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Testing was conducted between 30 April 2009 and 11 May 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD TG Draft Guideline (Version 7.6)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Guideline Testing of Chemicals B46
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19/08/09 Date of Signature: 04/03/09

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
ESponsor's identification : PDE-28
Description : red powder
Batch number : 1RF-8017
Date received : 23 March 2009
Storage conditions : room temperature in the dark

Test animals

Species:
other: reconstituted human epidermis model
Strain:
other: reconstituted human epidermis model
Details on test animals and environmental conditions:
Not applicable

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:

TEST MATERIAL

- Amount(s) applied (volume or weight with unit):
10 mg of the test material was applied to the epidermis surface.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 Minutes & 42 hour post exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
- Area of exposure:
10 mg of the test material was applied to the epidermis surface..

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.

- Time after start of exposure:
15 minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability):
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following table:
Criteria for in vitro interpretation Classification
Mean tissue viability is ≤50% Irritant (I) R38
Mean tissue viability is >50% Non-Irritant (NI)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
96.5
Remarks on result:
other:
Remarks:
Basis: mean Viability of cells. Time point: Day 6. Max. score: 96.5. Reversibility: other: Not applicable. Remarks: EPISKINTM RECONSTITUTED HUMAN EPIDERMIS MODEL. (migrated information)

In vivo

Irritant / corrosive response data:
Not applicable
Other effects:
No

Any other information on results incl. tables

Direct MTT Reduction:

The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material:

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 96.5% after a 15‑minute exposure.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared blue which was considered indicative of viable tissue.

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was≤ 40% relative to the negative control treated tissues and the Standard Deviation (SD) value of the % viability was ≤ 20%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was ≥ 0.6 and the SD value of the % viability was ≤ 20%. The negative control acceptance criterion was therefore satisfied.

Table1:              Mean OD540 Values and % Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

OD540 of tissues

Mean OD540 of triplicate tissues

± SD of OD540

Relative individual tissue viability %

Relative mean % viability

± SD of % viability

Negative Control Material

0.910

0.878

0.03

103.6

100*

3.4

0.875

99.7

0.850

96.8

Positive Control Material

0.242

0.238

0.03

27.6

27.1

2.9

0.211

24.0

0.261

29.7

Test Material

0.853

0.847

0.03

97.2

96.5

3.5

0.814

92.7

0.875

99.7

 


*= The mean viability of the negative control tissues is set at 100%

Table2              Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Tissue 1

Tissue 2

Tissue 3

Negative Control Material

-

-

-

Positive Control Material

++

++

++

Test Material

-

-

-

MTT visual scoring scheme
-
          =         blue tissue (viable)
+
         =         blue/white tissue (semi-viable)
++
       =         tissue is completely white (dead)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The test material was considered to be Non-Irritant to the skin.
Executive summary:
Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end‑point will be used to either confirm a non-irritant result or will be used to override the non‑irritant result.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96‑well plate. The optical density was measured at 540 nm.

Data are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).


Results: The relative mean viability of the test material treated tissues was 96.5% after a 15‑minute exposure.

Quality criteria:  The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: The test material was considered to be non-irritant to the skin.