Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2013 - 29 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: EU Method B.46 (In vitro dermal irritation)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
other: OECD Guideline 439 (In vitro dermal irritation)
Deviations:
no
Principles of method if other than guideline:
§ Test for direct MTT reduction with the test item: due to a typing error in the study plan, the volume of MTT solution used for the negative control in the preliminary test for direct MTT reduction which is specified in the study plan is incorrect. Indeed, the volume of MTT solution used in this study was 2 mL (as described in CiToxLAB’s SOP) instead of 2.2 mL. This deviation was considered not to have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
no

In vitro test system

Test system:
human skin model
Source species:
other: reconstructed human epidermis
Details on test system:
EpiskinTM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.
Medium and Incubation T°C: 37°C
REMOVAL OF TEST SUBSTANCE
- Rinsing: At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items. Excess D-PBS was removed by blotting the bottom of the tissue culture insert with absorbent paper.
The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 (± 1) hours.

POSITIVE CONTROL
Name: Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution.
The supplier and batch number of the positive control used are documented in the study files.

NEGATIVE CONTROL
Name: Dulbecco’s Phosphate-Buffered Saline (D-PBS).
The supplier and batch number of the negative control used are documented in the study files.

SCORING SYSTEM:
- Optical density (OD) was measured at 570 nm:
Relative mean viability (%) = 100 x mean cOD(test item) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank

Interpretation: see in "any other information on materials and methods"
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
Amount(s) applied per tissue: 10 µL ± 2 µL
Duration of treatment / exposure:
Exposure period of 15 minutes, followed by a rinsing.
Duration of post-treatment incubation (if applicable):
MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells, after 3-hour incubation.
Number of replicates:
Triplicate tissues for each tested substances (test item, negative control, positive control).

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
112
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

Main test
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.
 
Following a 15 -minute exposure and a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 112% with a standard deviation of 6%. As the mean viability was > 50% after the MTT reduction,the results met the criteria for an in vitro classification as non-irritant to skin.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is considered to be non-irritant to skin.
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTMreconstructed human epidermis model.

The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB’s standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

 

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were topically applied on triplicate tissues and incubated at room temperature for 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) hours at, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

Results

 

Preliminary tests

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential.

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

 

Following a 15 -minute exposure and a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 112% with a standard deviation of 6%. As the mean viability was > 50% after the MTT reduction,the results met the criteria for an in vitro classification as non-irritant to skin.

 

Conclusion

 

Under the experimental conditions of this study, the irritancy potential of the test item is considered to be non-irritant to skin.