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Description of key information

Skin Irritation

In an in vitro OECD 439 study, under GLP conditions, using EPISKIN™ Reconstructed Human Epidermis Model the test item is considered not irritating to skin.

Skin Corrosion

In an in vitro OECD 431 study, under GLP conditions, using EpiDerm™ Human Skin Model the test item is considered not corrosive to skin.

Eye Irritation

In an in vitro OECD 437 study, under GLP conditions, using Bovine Cornea the test item is considered not irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 May 2016 to 10 May 2016
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
Remarks:
EPISKIN™ reconstructed human epidermis
Source species:
human
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 16-EKIN-018
- Delivery date: 03 May 2016
- Date of initiation of testing: 05 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: no reference filter

NUMBER OF REPLICATE TISSUES: three per treatment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure period followed by the 42-Hour post-exposure incubation period is less than 50%
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure period followed by the 42-Hour post-exposure incubation period is equal to or more than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl Sodium Dodecyl Sulphate
- Concentration (if solution): 5% v/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
three per treatment
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue Sample 1
Value:
95.2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue Sample 2
Value:
103.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue Sample 3
Value:
104.4
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean (of 3 test tissue samples)
Value:
101.1
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No. Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert and also that the temperature indicator colour and agar medium colour were satisfactory so as to confirm the tissues were acceptable for use in the study. The tissue model supplier also demonstrated the batch of tissues was acceptable by determining the barrier function and also performed histological examination demonstrating appropriate morphology of the tissues.
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple. Therefore the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colourless. It was therefore unnecessary to use additional colour correction tissue controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%. The mean OD562 for the negative control treated tissues was 0.730 and the standard deviation value of the viability was 5.0%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%. The relative mean tissue viability for the positive control treated tissues was 7.9% relative to the negative control treated tissues and the standard deviation value of the viability was 4.4%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.1%. The test item acceptance criterion was therefore satisfied.

The mean OD562values and viabilities for the negative control, positive control and test item are presented in the table below:

 Item  OD562 of tissues  Mean OD562 of triplicate tissues

 ± SD of OD562

 Relative individual tissue viability (%)

 Relative mean viability (%)

  ± SD Relative mean viability (%)

 Negative Control Item

 

 

 

 

 0.701

 0.730

 

 

 

 

 0.037

 

 

 

 

 N/A

 100*

 

 

 

 

 N/A

 

 

 

 

 0.718

 N/A

 0.771

 N/A

 Positve Control Item      

 0.072

 0.058      

 0.032      

 9.9

 7.9      

 4.4      

 0.022  3.0
 0.081  11.1
 Test Item        0.695  0.738        0.037        95.2  101.1        5.1      
 0.757  103.7
 0.762  104.4

* The mean viability of the negative control tissues is set at 100%

OD = Optical Density

SD = Standard deviation

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this EPISKIN(TM) assay according to OECD Guideline 439, the results indicate that the test item is non-irritant to the skin.
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN(TM) reconstructed human epidermis model according to OECD Guideline 439. 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 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. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals from 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 a 96-well plate. The optical density was measured at 562 nm. Data was presented as percentage tissue viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of test item treated tissues was 101.1% compared to the negative control after the 15-minute exposure period and 42-hours post-exposure incubation period. This is above the 50% threshold for classification and therefore the test item was considered to be non-irritant to the skin under the conditions of this assay. The quality criteria required for acceptance of results in the test were met, therefore the study is considered valid.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 April 2016 to 14 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Remarks:
EpiDerm™ Human Skin Model
Source species:
human
Cell type:
other: Epithelial cells derived from human skin and formed into a stratified and cornified epithelium.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM ™
- Tissue batch number(s): 23328
- Delivery date: 12 April 2016
- Date of initiation of testing: 13 April 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 Microplate reader
- Wavelength: 562nm

NUMBER OF REPLICATE TISSUES: 2 per treatment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Distilled Water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL Potassium Hydroxide
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
3 and 60 minutes
Number of replicates:
Two tissues per treatment group for test item group and positive and negative controls.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean percent cell viabilites at three minute
Value:
100.6
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean percent cell viabilties at 60 minutes
Value:
103.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS
- Visible damage on test system: Not reported
- Direct-MTT reduction: No colour change was observed after one hour of incubation with the test item in MTT working solution, thus the test item did not react with MTT. Therefore, additional controls and data calculations were not necessary to account for the MTT-reducing potential of the test item.
- Colour interference with MTT: The solution containing the test item was a pale yellow colour. This colour was attributed to the intrinsic colour of the test item and was considered not to have the potential to cause colour interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 2.112 for the 3-minute exposure period and 2.105 for the 60-minute exposure period. The negative control acceptance criteria as per the OECD guideline were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 3.7% relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the coefficient of variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Mean OD562values and viabilities for the negative control, positive control and test item are given in the table below along with the relative mean cell viabilities for each treatment group.

 Tissue

 Exposure Period

(minutes)

 Mean OD562 of Individual Tissues  Mean OD562 of Duplicate Tissues  Standard Deviation  Coeffcient of Variation (%)  Relative Mean Viability (%)
 Negative Control           3     2.192  2.112     0.114     5.4     100*         
 2.031
 60     2.193  2.105     0.125     5.9   
 2.016
 Positive Control           3     0.073  0.069     0.006     N/A     3.3   
 0.065
 60     0.066  0.077     0.016     N/A     3.7   
 0.088
 Test Item           3     2.022  0.144     0.144     6.8     100.6   
 2.226
 60     2.139  0.054     0.054     2.5     103.4   
 2.215

*The mean % viability of the negative control tisue is set at 100%

N/A = not applicable

Interpretation of results:
GHS criteria not met
Conclusions:
The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model according to the OECD Guideline 431. The mean percent cell viability for the test item at 3 and 60 minutes was 100.6% and 103.4%, respectively, therefore under the conditions of this assay the test item was considered to be non-corrosive to the skin.
Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes, according to the OECD Guideline 431.

The corrosion potential of a test item is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to predict the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

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

The mean percent cell viability for the test item at 3 and 60 minutes was 100.6% and 103.4%, respectively, therefore under the conditions of this assay the test item was considered to be non-corrosive to the skin.

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:
26 May 2016
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:
SOURCE OF COLLECTED EYES
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were 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 prepared immediately on arrival.
Vehicle:
other: 0.9% w/v sodium chloride solution.
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% w/v

VEHICLE
- Concentration (if solution): 0.9% w/v
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three per treatment
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES
Three per treatment

NEGATIVE CONTROL USED
0.9% w/v sodium chloride solution

POSITIVE CONTROL USED
Imidazole, as a 20% w/v solution in 0.9% w/v sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of either the test item, negative, control or positive control were applied to the appropriate corneas for 240 minutes.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three rinses with Eagle's Minimum Essential Medium
- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in corneal opacity (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG were used
Irritation parameter:
in vitro irritation score
Run / experiment:
Test Item
Value:
0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Measured at 240 minutes.
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative Control
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Measured at 240 minutes.
Irritation parameter:
in vitro irritation score
Run / experiment:
Positive control
Value:
94.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Measured at 240 minutes.
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The positive control IVIS (94.7) was within the historical control range for the laboratory and thus the positive control acceptance criterion was satisfied.
- Acceptance criteria met for positive control: The negative control gave a mean opacity of 1.7 and a mean permeability 0.019 and was within the historical control for the laboratory and thus the negative control acceptance criteria were satisfied.



Interpretation of results:
GHS criteria not met
Conclusions:
The eye irritation/ corrosivity potential of the test substance was evaluated in a test according to the OECD Guideline 437. The in vitro irritancy score (IVIS) score for the test substance at 240 minutes was 0.1. Therefore, the test substance was found to be non-irritant therefore not assigned a UN GHS classification category.
Executive summary:

The purpose of this test was to evaluate the eye irritation/ corrosivity potential of the test substance according to the OECD Guideline 437. The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain 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 is used to identify test items (both chemicals and mixtures) which are potential serious eye corrosives or irritants as well as those which are non-irritant, 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 that are non-irritants 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 test substance was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes to the surface of the corneas. 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 score for the test substance at 240 minutes was 0.1. IVIS scores for the negative and positive control were 2.0 and 94.7, respectively. Therefore, the test substance was found to be non-irritant and was not assigned a UN GHS classification category.

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

Additional information

Justification for classification or non-classification

Based on the reliable and valid in vitro studies on skin corrosion (OECD TG 431) and skin irritation (OECD TG 439), the substance does not require a classification for skin corrosion/skin irritation in accordance with Regulation (EC) No. 1272/2008. Based on the reliable and valide in vitro study on eye irritation (OECD TG 437), the substance does not require a classification for eye irritation in accordance with Regulation (EC) No. 1272/2008.