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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed in a GLP laboratory in accordance with OECD and EU guidelines with minor deviations that were not considered not to affect the purpose or integrity of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The control groups for this study were shared with study 41402573. The control groups raw data will be filed with study 41402564.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
The control groups for this study were shared with study 41402573. The control groups raw data will be filed with study 41402564.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Off white solid blocks

Test animals

Species:
other: SKINETHIC™ Reconstructed Human Epidermis Model

Test system

Type of coverage:
open
Preparation of test site:
not specified
Controls:
other: SKINETHIC™ Reconstructed Human Epidermis Model
Amount / concentration applied:
16 mg of the test itemwas applied topically to the corresponding tissues ensuring uniform covering. 10 μL of sterile water was topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis.
Duration of treatment / exposure:
42 minutes
Observation period:
42 hours
Number of animals:
Quadruplicate tissues were treated with test item, negative and positive controls.
Details on study design:
TEST SITE
- Area of exposure: 0.5 cm2

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 DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the 6-well post exposure incubation plates prefilled with 2.0 mL of growth medium.
- Time after start of exposure: 42 minutes

SCORING SYSTEM:

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
ca. 100
Remarks on result:
other:
Remarks:
Basis: mean Negative control. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
ca. 1.2
Remarks on result:
other:
Remarks:
Basis: mean Positive control. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
ca. 56.9
Remarks on result:
other:
Remarks:
Basis: mean Test item. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)

Any other information on results incl. tables

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

 

 

Item

 

OD562 of tissues

 

Mean OD562 of triplicatet issues

 

±SD of

OD562

Relative individual tissue viability (%)

 

Relative mean viability (%)

± SD of Relative mean viability (%)

 

Negative

Control

Item

2.166

 

 

 

2.039

 

 

 

0.111

106.2

 

 

100

 

 

 

5.4

 

1.961

 

96.2

 

1.989

 

97.5

 

 

Positive

Control

Item

 

0.029

 

 

 

0.025

 

 

 

0.003

 

1.4

 

 

 

1.2

 

 

 

0.2

 

0.024

 

1.2

 

0.023

 

1.1

 

 

 

TestItem

 

1.118

 

 

 

1.159

 

 

 

0.038

 

54.8

 

 

 

56.9

 

 

 

1.9

 

1.192

 

58.5

 

1.168

 

57.3

 

 

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was classified as non-irritant. The following classification criteria apply:
EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the SKINETHICTM reconstructed human epidermis model after a treatment period of 42 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric 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 item treated tissues relative to the negative controls. Histopathological analysis maybe conducted if considered necessary. The concentration of the cytokine or LDH release in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined under certain circumstances.

Quadruplicate tissues were treated with the test item or control items for an exposure period of 42 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period three tissues for each group were taken for MTT-loading. The remaining treated tissues were retained for possible histology. The growth medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading the inserts were removed and immersed in 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 triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm.

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

The relative mean viability of the test item treated tissues was 56.9% after the 42-Minute exposure period and 42 hour post-exposure incubation period. It was considered unnecessary to proceed with tissue histology or analysis of inflammatory mediators.

The test item was classified as non-irritant. The following classification criteria apply: EU DSD & CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).