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Description of key information

For the test substance negative in vitro studies on skin and eye irritation and skin corrosion are available. For the  analogue negative in vivo studies are available for skin and eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-04-2012 to 17-04-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according to the guideline and under GLP
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Principles of method if other than guideline:
exposure period 15 minutes with post-exposure incubation period of 42 hours
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Details on study design:
The EpiSKIN assay uses reconstructed human epidermis to assess skin irritation. For the viability test enzymatic conversion of MTT to formazan is used.

In a pre-test direct interaction of the test substance with the detection chemical MTT is assessed.
In the main study triplicate tissues were treated with 30 uL of the test substance during 15 minutes. In addition a negative control (DPBS) and a positive control (5% SDS) were included. After treatment tissues were rinsed, incubated in maintenance fluid for 42 hours at 37 °C and treated with MTT during 3 hours (at 37 °C). After MTT loading a total biopsy of each epidermis (epidermis and collagen) was made, that was suspended in acidified iso-propanol for 3 days and kept in the refrigerator at 1-10°C. Thereafter the optical density of the extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control.
Tissue viability > 50% means non-irritant
Tissue viability ≤ 50% means irritant
Irritation / corrosion parameter:
% tissue viability
Value:
78.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
6.0% tissue viability
Other effects:
The test substance did not interfere with the reduction of MTT to blue formazan salt.
In the main test after rinsing, the test substance was found to remain on the tissue surface due to the extreme viscosity.

 

Mean OD540 ± SD

Relative mean viability ± SD

Negative control:

0.883 ± 0.087

100 ± 9.8%

Test substance:

0.675 ± 0.085

78.6 ± 8.0%

SDS:

0.053 ± 0.018

6.0 ± 2.1%

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: tissue viability > 50%: non-irritant
Conclusions:
The test substance is non-irritant in the EPISKIN reconstructed human epidermal model.
Executive summary:

In this assay triplicate reconstructed human skin tissues were treated with 30 uL of the test substance during 15 minutes. After treatment tissues were rinsed, kept for 42 hours and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control. The viability was 78.6%, which is indicative for non-irritant substances.

The test substance is non-irritant in the EPISKIN reconstructed human epidermal model.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-05-2012 to 17-05-2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: not formally validated study method
Principles of method if other than guideline:
The SkinEthic reconstructed human corneal epithelium assay is used
GLP compliance:
yes (incl. certificate)
Details on study design:
The SkinEthic reconstructed human corneal epithelium assay uses corneal epithelial tissue to assess penetration and concomittant cytotoxicity of the test substance.

In a pre-test direct interaction of the test substance with the detection chemical MTT is assessed.
In the main study triplicate SkinEthic tissues were treated with 30 uL of the test substance during 10 minutes. In addition a negative control (Na2HPO4 0.142 g/L; glucose 1.802 g/L; HEPES 7,149 g/L; KCl 0.224 g/L; NaCl 7.597 g/L) and a positive control (2% SDS) were included. After treatment tissues (2 per treatment) were rinsed and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control.
Tissue viability ≥ 60% means non-irritant
Tissue viability < 60% means irritant
The third tissue per treatment was preserved for histopathology.
Irritation parameter:
other: % tissue viability
Value:
79
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
25.5% tissue viability
Other effects:
The test substance did not interfere with the reduction of MTT to blue formazan salt.
In the main test after rinsing the test substance was found to remain on the tissue surface due to the extreme viscosity.

 

Mean OD540

Relative mean viability

Negative control:

0.729

100%

Test substance:

0.626

79%

SDS:

0.202

25.5%

No histopathology was performed.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: < 60% viability is indicative for positive result
Conclusions:
The test substance is non-irritant in the SkinEthic reconstructed human corneal epithelium assay.
Executive summary:

In this assay triplicate SkinEthic tissues were treated with 30 uL of the test substance during 10 minutes. After treatment tissues (2 per treatment) were rinsed and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. Viability was expressed as percentage MTT conversion versus negative control. The viability was 79%, which is indicative for non-irritant substances.

As the assay is not formally validated, the results need to be regarded as preliminary.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

In the EPISKIN assay for irritation, triplicate reconstructed human skin tissues were treated with 30 uL of the test substance during 15 minutes. After treatment tissues were rinsed, kept for 42 hours and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. The viability was 78.6%, which is indicative for non-irritant substances (Harlan 2012h).

In the EpiSKIN test for corrosion, duplicate reconstructed human skin tissues were treated with 30 uL of the test substance during 3, 60 and 240 minutes. After treatment tissues were rinsed and incubated with MTT for 3 hours (at room temperature). Thereafter tissues were allowed to dry, epidermis and collagen were separated, suspended (after vortexing) in isopropanol and kept overnight (all procedures in dark). Optical density of homogene solutions of MTT treated tissues was measured at 540 nm. The viability was 86.1 -97.5% for the different exposure times, which is indicative for non-corrosive substances (Harlan 2012i).

In an in vivo test in rabbits with an anlogue substance exposure of the skin for 4 hours lead to minimal erythema and oedema after 1, 24 and 48 hours. At the 72 hour observation the effects were fully reversed. Therefore the analogue is concluded to be not irritating to the skin (A Marxer 1999a).

Eye irritation

In SkinEthic assay tissues (in triplicate) were treated with 30 uL of the test substance during 10 minutes. After treatment tissues (2 per treatment) were rinsed and treated with MTT. Optical density of extracted MTT solutions was measured at 540 nm. The viability was 79%, which is indicative for non-irritant substances. As the assay is not formally validated, the results need to be regarded as preliminary (Harlan 2012j).

In an in vivo test in rabbits no effects on cornea and iris and very limited effects on the conjunctivae were reported after application of an analogue substance. The effects were fully reversible and therefore it is concluded that this substance is not an eye irritant (A Marxer 1999b).

In a weight of evidence approach it is concluded that the available data are sufficient to conclude that the test substance is not irritating to skin and eyes. The negative in vitro data with the test substance are confirmed by the in vivo data with the analogue. The rationale for the read-across can be found in chapter 13.


Justification for selection of skin irritation / corrosion endpoint:
The outcome of the in vitro study with the test substance is confirmed by the in vivo study with an analogue (Marxer 1999a).

Justification for selection of eye irritation endpoint:
The outcome of the in vitro study with the test substance is confirmed by the in vivo study with an analogue (Marxer 1999b).

Justification for classification or non-classification

The test substance does not need to be classified for skin and eye irritation.