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Description of key information

OECD439: In vitro Skin Irritation test using the human skin model.

The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS). Depending on the regulatory framework and applicability of the test guideline, chemicals that produce cell viabilities above the defined threshold level, are considered non-irritants. The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.

A skin corrosion study according to OECD Guideline 431 has been initiated. The corresponding study plan is available. The final report will be provided in August 2018 and the result will be submitted afterwards.

OECD437: Bovine Corneal Opacity and Permeability assay.

This test is designed to measure the opacity of the cornea by quantifying the ability of light to pass through. The permeability, as a result of the irritation potential of the test item, is determined using Na-fluorescein solution. The comparison of the opacity before and after the exposure to the test item and the determination of the permeability after the treatment provide an indication of the damaging effect of the test item.

For this purpose the induction of opacity and increased permeability in an isolated bovine cornea after application of the test item was measured. The results of both criteria were combined. Under the reported experimental conditions the test item did not cause serious eye damage (Category 1: CLP/GHS). However, the study is not sufficient to be used for hazard classification under the CLP regulation.

A reconstructed human cornea-like epithelium test according to OECD Guideline 492 has been initiated. The corresponding study plan is available. The final report will be provided in August and the result will be submitted afterwards.

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:
08 February 2018 to 23 March 2018
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)
Version / remarks:
(2015)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Reconstructed epidermis of normal human keratinocytes, originating from adult donors
Source strain:
other: Keratinocyte strain: 00267
Justification for test system used:
The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin irritation potential of a test item by assessment of its effect on a three dimensional human epidermis model.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM and EPIDERMTM)
- Tissue batch number(s): 25888
- Expiration date: Not specified
- Shipping date: March 20th 2018
- Date of initiation of testing: March 20th 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): All incubations were carried out at 37.0 ± 1.5⁰ C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with DPBS to
remove residual test item after exposure period.
- Observable damage in the tissue due to washing: Not specified.
- Modifications to validated SOP: The tissues were rinsed with PBS instead of DPBS to remove any residual material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: (1 mg/mL)
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices) .
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- No. of replicates: 3

PREDICTION MODEL / DECISION CRITERIA
-The test substance is considered to be an irritant to skin if the relative mean tissue viability after a 15-minute exposure period followed by the 42-Hour post-exposure incubation period is less than 50% of the mean viability of the negative controls.
-The test substance is considered to be non-irritant to skin if the relative mean tissue viability after a 15-minute exposure period followed by the 42-Hour post-exposure incubation period is greater than 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 +/- 2mg (approx. 39 mg/cm2) wettd with 25µL DPBS
- Concentration (if solution): Not applicable.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25µL
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL Sodium dodecyl sulphate
- Concentration (if solution): 5 %
Duration of treatment / exposure:
15 Minutes
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37°C.
Number of replicates:
3 test replicates.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
4.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
3.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
7.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- Acceptance criteria met for negative control: The negative control value from the study had a mean relative tissue viability of 100% and a mean OD570 of 1.718 which is within the historic control data
range.
- Acceptance criteria met for positive control: The mean relative tissue viability of the positive control was ≤50% (2.9%) and the mean OD570 was 0.051 which is within the historic control data range.
- Acceptance criteria met for variability between replicate measurements: The standard deviation for the negative control was within the acceptance criteria of ≤18 for the percentage viability (8.5%). The standard deviation for the positive control was within the acceptance criteria of ≤18 for the percentage viability (0.1%). All validity criteria were met.

Table 1- Results of treatment with test item and controls

Treatment Group Tissue number   OD 570 nm well 1   OD 570 nm well 2   OD 570 nm well 3 Mean OD of 3 wells Mean OD of 3 wells blank corrected  Mean OD of 3 tissues blank corrected     Rel. viability [%] Tissue 1,2 +3*   Standard deviation Mean Rel. viability [%]**
 Blank   0.039  0.038  0.038  0.038           

Negative

control

1

2

3

1.947

1.668

1.750 

1.888

1.598

1.717 

1.908

1.614

1.714 

1.914

1.627

1.727 

1.876

1.589

1.689 

 1.718

109.2

92.5

98.3 

8.5

 

100.0

 

Postive control

1

2

0.089

0.091

0.088 

0.088

0.088

0.086 

0.088

0.093

0.087 

0.089

0.091

0.087 

0.050

0.053

0.049 

0.051

 

2.9

3.1

2.8 

0.1   2.9
 Test item

1

2

0.090

0.097

0.177 

0.090

0.095

0.171 

0.090

0.096

0.174 

0.090

0.096

0.174 

0.052

0.058

0.136 

0.082

 

3.0

3.4

7.9 

 2.7  4.8
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In conclusion, the test item is a skin irritant according to CLP (H315) and GHS (category 2).

Executive summary:

The study is assigned a reliability score of 1 (reliable without restrictions) as it followed OECD Guideline 439: In Vitro Skin irritation: Reconstructed Human Epidermis (RHE) Test and it is compliant with GLP. 25 +/-2 mg of the test item (approx 39 mg/cm2 according to guideline) was wetted with 25 µL of DPBS and applied to the EpiSkin tissue for 15 minutes followed by a 42-hour post-exposure incubation period. Negative and positive controls were also tested. The positive control had a mean cell viability of 2.9%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 7%.

Therefore, all validity criteria were met. The substance under test conditions had a mean tissue viability of 4.8% compared to the negative control. This value is below the threshold for irritancy (50%). This concludes the test item is a skin irritant (Category 2).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
20 February 2018 to 02 March 2018
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)
Version / remarks:
(2017)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
(2010)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- Source: Slaughterhouse (AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany)
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): 9-month-old cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in HBBS containing 1 % (v/v) Penicillin/Streptomycin in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Not specified
- indication of any existing defects or lesions in ocular tissue samples: All eyes were checked for unacceptable defects by removing them from the storage solution and examining them. Those exhibiting defects were discarded.
- Indication of any antibiotics used: The cattle eyes were stored in HBSS containing 1% Penicillin/Streptomycin during transportation.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml was introduced onto the epithelium of the cornea.
- Concentration (if solution): The test material was prepared as a 20 % suspension (w/v) in saline.

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% NaCl
- Lot/batch no. (if required): Not specified
- Purity: Not specified
Duration of treatment / exposure:
The test consisted of the application of the 20 % (w/v) suspension in saline of the test item to the fresh bovine cornea; followed by an initial opacity measurement. After exposure, the cornea was thoroughly rinsed to remove the test item and incubated for 240 minutes in incubation medium. After incubation in the medium, opacity measurements and permeability values were determined after a 90 minutes incubation period in sodium fluorescein.
Duration of post- treatment incubation (in vitro):
90 +- 5 minutes at 32 +-1°C
Number of animals or in vitro replicates:
Three test replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Preparation: The eyes were checked for defects such as opacity, scratches, pigmentation and vascularization by removing them from the HBSS containing 1 % (v/v) Penicillin/Streptomycin.


QUALITY CHECK OF THE ISOLATED CORNEAS : All eyes were checked macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

NUMBER OF REPLICATES : Three

NEGATIVE CONTROL USED : Physiological saline

SOLVENT CONTROL USED (if applicable) : Not applicable
POSITIVE CONTROL USED : 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline)

APPLICATION DOSE AND EXPOSURE TIME : 0.75 ml was introduced onto the epithelium of the cornea.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: 90 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Not specified
- POST-EXPOSURE INCUBATION: 240 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a Opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Not specified

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
In vitro score range UN GHS
≤ 3 No Category
> 3 ≤ 55 No prediction can be made
>55 Category 1


DECISION CRITERIA: The decision criteria for the IVIS cut-off values for identifying test chemicals as inducing serious eye damage was used for the study.
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item Mean
Value:
21.33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not applicable

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The first negative control value was outside the acceptance criteria range of 0.86 – 1.64. Due to this, the experiment was repeated twice. The overall IVIS mean for the negative control was 1.03, which was within the acceptance criteria range.
- Acceptance criteria met for positive control: The positive control value of 108.55 was within the acceptance criteria range of 98.30 – 138.03.

Table 1: results after 240 minutes exposure period.

Test Group

Opacity value = Difference

(t240-t0) of Opacity

 

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

      1.03

 

No category

Negative control

1

 

0.33

0.045

 

0.046

1.68

0

0.044

0.66

0

0.050

0.75

Positive control

95.67*

0.627*

105.07

 

108.55

 

Category 1

103.67*

0.735*

114.69

10.67*

0.483*

105.91

Test item

8.67*

0.964*

23.12

 

 

21.33

No prediction can be made

7.67*

0.880*

20.86

10.67*

0.623*

20.01

Interpretation of results:
study cannot be used for classification
Remarks:
Not corrosive or a severe irritant to the eye but study is not sufficient for hazard assessment
Conclusions:
In conclusion, under the reported experimental conditions the test item did not cause serious eye damage (Category 1: CLP/GHS). However, the study is not sufficient to be used for hazard classification under the CLP regulation.
Executive summary:

The study is a GLP-compliant study following OECD guideline 437 and is therefore reliable without restrictions (Klimisch 1).  A 20 % (w/v) suspension in saline of the test item was applied directly on top of the corneas. The positive control mean in vitro irritancy score of 108.55 was within two standard deviations of the current historical positive control mean. The negative control values for opacity and permeability (1.03) were less than the upper limits of the laboratory historical range. The validity criteria for the study was therefore met. The test item had an In vitro Irritancy score of 21.33. Based on this result the test item did not cause serious eye damage (Category 1: CLP/GHS); however, this study is not sufficient to be used for hazard classification under CLP regulation.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean relative viability value decreased to 4.8% compared to the relative absorbance value of the negative control.

Eye irritation:

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 21.33.

Justification for classification or non-classification

Skin irritation:

Since the mean relative viability value decreased to 4.8% compared to the relative absorbance value of the negative control, this value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

A skin corrosion study according to OECD Guideline 431 has been initiated. The corresponding study plan is available. The final report will be provided in August 2018 and the result will be submitted afterwards.

Eye irritation:

As the calculated mean in vitro irritancy score was 21.33, according to OECD 437 guideline criteria the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1) but the test item’s hazard for eye damaging cannot be predicted.

A reconstructed human cornea-like epithelium test according to OECD Guideline 492 has been initiated. The corresponding study plan is available. The final report will be provided in August and the result will be submitted afterwards.