Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
other: reconstituted human epidermis model

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
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
Observation period:
Not applicable
Number of animals:
Not applicable
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:
expressed as mean relative absorbance
Run / experiment:
all 3
Value:
> 97.8 - < 105.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Results after treatment with the test item and the controls

 

Dose Group

Ex-po-sure Interval

Tissue No.

Absorbance 570 nm
Well 1

Absorbance 570 nm
Well 2

Absorbance 570 nm
Well 3

Mean Absorbance of 3 Wells

Mean-Absorbance

of three wells blank

corrected

Mean

Absorbance

of 3 wells

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

0.000

 

 

 

 

 

 

Negative Control

60 min

Tissue 1

1.736

1.703

1.695

1.711

1.675

1.673

100.1

1.1

100.0

 

 

Tissue 2

1.679

1.688

1.699

1.689

1.653

98.8

Tissue 3

1.725

1.713

1.740

1.726

1.690

101.0

Positive Control

60 min

Tissue 1

0.116

0.116

0.118

0.117

0.081

0.075

4.8

6.6

4.5

 

 

Tissue 2

0.107

0.111

0.109

0.109

0.073

4.4

Tissue 3

0.108

0.105

0.110

0.107

0.071

4.3

Test Item

60 min

Tissue 1

1.800

1.774

1.835

1.8037

1.767

1.711

105.7

4.0

102.3

0.009

101.8

Tissue 2

1.677

1.685

1.654

1.672

1.636

97.8

Tissue 3

1.765

1.758

1.775

1.766

1.730

103.4

 

* relative absorbance per tissue [rounded values]: 100 x (absorbancetissue) divided by mean absorbance negative control

** relative absorbance per treatment group [rounded values]: 100 x (mean absorbance test item/positive control) divided by (mean absorbance negative control)


Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
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 OD³0.8 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 % variability values 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 not reduced (101.8%) 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.