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Description of key information

In vitro testing was conducted in order to investigate the irritancy potential of the substance to the skin and the eye.

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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
10 µL (26.3 µL/cm2) of the test material was applied to the epidermis surface
Duration of treatment / exposure:
exposure period of 15 minutes
Duration of post-treatment incubation (if applicable):
42 Hour post-exposure incubation period
Irritation / corrosion parameter:
% tissue viability
Value:
94.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-irritant.
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 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.


Triplicate tissues were treated with the test item for an exposure period of 15 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 each tissue was taken for MTT-loading. The maintenance 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 a total biopsy of each epidermis was made and placed into micro tubes containing acidified 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 duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 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 94.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.


The quality criteria required for acceptance of results in the test were satisfied.


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

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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes
Species:
cattle
Details on test animals or tissues and environmental conditions:
Placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.
Vehicle:
Hank's balanced salt solution
Amount / concentration applied:
0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Three corneas were also allocated to the test item.
Three corneas to the positive control item.
Three corneas to the negative control item.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

SCORING SYSTEM:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

IVIS Classification
<= 3 No category. Not requiring classification
>3; <=55 No prediction of eye irritation can be made
>55 Category 1, UN GHS or EU CLP Causes serious eye damage

TOOL USED TO ASSESS SCORE: Camspec Model M108 Spectrophotometer.
Irritation parameter:
in vitro irritation score
Value:
0.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The test material was classified as non-irritant.
Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.
The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.


The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).


The IVIS of the test item is 0.9. According to the data interpretation criteria, no classification is required since the IVIS is ≤ 3.


Not requiring classification to UN GHS or EU CLP.

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

Additional information

In vitro skin irritation
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 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.
Triplicate tissues were treated with the test item for an exposure period of 15 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 each tissue was taken for MTT-loading. The maintenance 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 a total biopsy of each epidermis was made and placed into micro tubes containing acidified 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 duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 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 94.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
The quality criteria required for acceptance of results in the test were satisfied.
The test item was classified as non-irritant. The following classification criteria apply: EU CLP Not classified for Irritation.


 


In vitro eye irritation
The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.
The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.
The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).
The IVIS of the test item is 0.9. According to the data interpretation criteria, no classification is required since the IVIS is ≤ 3.
Not requiring classification to UN GHS or EU CLP.

Justification for classification or non-classification

The test substance was not considered as irritant when tested in vitro for its irritancy potential to the skin and the eye. Therefore, it does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008.