Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 January 2016 until 01 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 439 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:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: UN GHS (2003, last rev. 2009)
Deviations:
no
Principles of method if other than guideline:
Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL of DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
DPBS was used as negative control. 30 µL were applied to each of triplicate tissues for 1 hour.

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
A 5% SLS solution in deionised water was used as positive control. 30 µL were applied to each of triplicate tissues for 1 hour.
Duration of treatment / exposure:
60 minutes

Test animals

Species:
other: reconstituted human epidermis model

Test system

Type of coverage:
other: Topical
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL of DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.
Duration of treatment / exposure:
60 minutes
Details on study design:
Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative and the positive control for 60 minutes.
Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.
30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following 69.75 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
Episkin model, 3 tissues per concentration
Run / experiment:
mean
Value:
80.5
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
No other effects were detected

Any other information on results incl. tables

Results after treatment with Aluminium-Braun 1134 p and the controls (60 minutes exposure interval)


Dose Group Tissue
No.
Absorb.
570 nm
Well 1
Absorb.
570 nm
Well 2
Absorb.
570 nm
Well 3
Mean
Absorbance
of 3 Wells
Mean Absorbance
of 3 Wells
blank
corrected
Mean
Absorbance*
after blank
correction
Rel.
Absorbance
[%] Tissue
1, 2 + 3*
Relative Standard Deviation
[%]
Mean Rel.
Absorbance
[% of Negative Control]**
Mean Absorbance
Blank corrected viable tissue
without MTT
(Step 2)
Mean Rel.
Absorbance
[% of Negative Control]
after colour interference and MTT reduction correction
Blank   0.036 0.037 0.036 0.036 0            
Negative
Control
1 1.736 1.703 1.695 1.711 1.675 1.673 100.1 1.1 100    
2 1.679 1.688 1.699 1.689 1.653 98.8
3 1.725 1.713 1.74 1.726 1.69 101
Positive
Control
1 0.116 0.116 0.118 0.117 0.081 0.075 4.8 6.6 4.5    
2 0.107 0.111 0.109 0.109 0.073 4.4
3 0.108 0.105 0.11 0.107 0.071 4.3
Test
 Item
1 1.322 1.323 1.34 1.328 1.292 1.36 77.2 6.4 81.3 0.013 80.5
2 1.483 1.484 1.515 1.494 1.458 87.2
3 1.363 1.334 1.402 1.366 1.33 79.5

*          relative absorbance per tissue [rounded values]

**        relative absorbance per treatment group [rounded values]


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item passed the MTT interference pre-test. Due to its intensive colour, an additional test with one viable tissue (without MTT addition) was necessary to correct the result in the main experiment.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.5% thus ensuring the validity of the test system.

The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 7% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 80.5% (corrected value) after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.