Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 June 2008 and 01 July 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EPISKINTM reconstituted human epidermis model
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: The EPISKIN model is a three-dimensional reconstituted human epidermis model
Strain:
other: The EPISKIN model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiat
Details on test animals and environmental conditions:
TEST ANIMALS: Not applicable
TEST ANIMALS: Not applicable
- Tissue: The EPISKIN model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13 day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.




ENVIRONMENTAL CONDITIONS
- Temperature (°C): warmed to approximately 37ºC
- Humidity (%): Not applicable
- Air changes (per hr): Not applicable
- Photoperiod (hrs dark / hrs light): Not applicable


IN-LIFE DATES: Not applicable

Test system

Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):10 ± 2 mg of the test material was applied to the epidermis surface.
- Concentration (if solution): Not applicable


VEHICLE
- Amount(s) applied (volume or weight with unit):Not applicable
- Concentration (if solution): Not applicable
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable
Duration of treatment / exposure:
Triplicate tissues were treated with the test material for an exposure period of 15 minutes.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
- Area of exposure:Not applicable
- % coverage: Not applicable
- Type of wrap if used: Not applicable


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 with 25 ml of PBS. Rinsing was achieved by filling and emptying each tissue insert with PBS using a pipette. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37ºC, 5% CO2 in air for approximately 42 hours.
- Time after start of exposure: 15 minutes


SCORING SYSTEM: For each 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).

Criteria for in vitro interpretation
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: other: % Viabiltiy
Value:
102.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Max. score: 110.0. Reversibility: no data. Remarks: The relative mean viability of the test material treated tissues was 102.3% after a 15 minute exposure.. (migrated information)

In vivo

Irritant / corrosive response data:
According to the protocol the test material was considered to be Non-Irritant.
Other effects:
Following the 15-minute exposure the test material treated tissues appeared blue which was considered indicative of viable tissue.

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 OD540values, standard deviations and tissue viabilities for the negative control, test material and positive control 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 102.3% 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 tissueswas≤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 OD540for the negative control treated tissueswas≥0.6 and the SD value of the % viability was ≤20%. The negative control acceptance criterion was therefore satisfied.

Table 1-Mean OD540Values and % Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

OD540of tissues

Mean OD540of triplicate tissues

± SD of OD540

Relative individual tissue viability

Relative mean % viability

± SD of % viability

Negative Control MaterialÅ

0.753

0.791

0.090

95.2

100*

11.4

0.894

113.0

0.727

91.9

Positive Control MaterialÅ

0.131

0.110

0.019

16.6

13.9

2.4

0.100

12.6

0.098

12.4

Test Material

0.826

0.809

0.070

104.4

102.3

8.9

0.870

110.0

0.732

92.5

 


Table 2-Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Tissue 1

Tissue 2

Tissue 3

Negative Control MaterialÅ

-

-

-

Positive Control MaterialÅ

++

++

++

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: EU
Conclusions:
The test material was considered to be Non-Irritant.
Executive summary:

Introduction. The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTMreconstituted 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 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.

Methods. 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 store 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 102.3% 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.