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Administrative data

Description of key information

Skin irritation study (OECD 439): not irritating to skin
Eye corrosion study (OECD 437): not corrosive to eyes
Eye irritation study (OECD 405): not irritating to eyes

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:
19-22 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with EU-Method B.46 resp. OECD guideline 439
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: human skin model EpiDermTM
Amount / concentration applied:
25 mg on average
Duration of treatment / exposure:
60 minutes
Number of animals:
3 epidermic tissue
Details on study design:
This in-vitro study was performed in order to evaluate the potential of SABOSTAT A 300 to evoke skin irritation in a human-skin-model.
The test consists of a topical exposure of the neat test item to a human reconstructed epi-dermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT (= 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), pre-sent in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreat-ed negative controls is used to predict skin irritation potential.
Irritation / corrosion parameter:
other: other: Optical Density percentage with respect to negative control
Value:
119.6
Remarks on result:
other:
Remarks:
Basis: mean. (migrated information)

Findings and Results

 Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

   Absorbance values blank isopropanol (OD at 570 nm)

Replicate

1

2

3

4

5

6

7

8

Mean

Absorbance

0.038

0.037

0.037

0.038

0.036

0.037

0.037

0.037

0.037

 

The absorbance values of negative control, test item and positive control are given in the following table:

    Absorbance Values negative control, test item and positive control (OD at 570 nm)

Designation

Measurement

Negative Control

SABOSTAT A 300

Positive Control

Tissue 1 

1

1.828

2.320

0.088

2

1.794

2.250

0.085

Tissue 2 

1

1.786

1.999

0.083

2

1.780

1.980

0.083

Tissue 3 

1

1.877

2.235

0.089

2

1.840

2.215

0.089

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table above. Mean and relative standard deviation (comparison of the 3 tissues) were also calculated.

  Mean Absorbance Values

Designation

Negative Control

SABOSTAT A 300

Positive Control

Mean – blank (tissue 1)

1.774

2.248

0.050

Mean – blank (tissue 2)

1.746

1.953

0.046

Mean – blank (tissue 3) 

1.822

2.188

0.052

Mean of the 3 tissues

1.781

2.130

0.049

Relative standard deviation
of the 3 tissues

2.2%

7.3%

6.2%

 


Comparison of Formazan Production

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

    % Formazan Production

Designation

SABOSTAT A 300

Positive Control

% Formazan production (tissue 1)

126.2%

2.8%

% Formazan production (tissue 2)

109.7%

2.6%

% Formazan production (tissue 3)

122.9%

2.9%

% Formazan production (mean)

119.6%

2.8%

 

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is considered as not skin irritant.
After the treatment, the relative absorbance values were increased to 119.6%. This value is well above the threshold for skin irritation (50%).
The optical density of the negative control (mean OD = 1.8) was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control induced a decrease in the relative absorbance as compared to the negative control to 2.8 % (required:  20%) for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item.
For these reasons, the result of the test is considered valid.
Executive summary:

3 tissues of the human skin model EpiDermTMwere treated with the substance for 60 min. On average, 25 mg of the solid test item (wetted with 25 µL DPBS-buffer) were applied to each tissue and spread to match the tissue size (0.63 cm2; as indicated by supplier). DPBS-buffer was used as negative control, 5 % SDS solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.8. The positive control showed clear irritating effects. Relative absorbance was reduced to 2.8 %. Variation within tissues was acceptable (< 18%).   After the treatment with the test item, the relative absorbance values were increased to 119.6%. This value is well above the threshold for irritation potential (50%).   Therefore,the substance is considered as not skin irritant in the Human Skin Model Test.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to international guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
other: OECD Guidelines for the Testing of Chemicals, adopted 25. Jul. 2014, DRAFT PROPOSAL FOR A NEW TEST GUIDELINE , Reconstructed Human Cornea-like Epithelium (RhCE)
Qualifier:
according to
Guideline:
other: “EpiOcular TM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irri-tation of chemicals” by MatTek Corporation, document no. MK‐24‐007‐0055, dated 10. Dec. 2012
GLP compliance:
yes (incl. certificate)
Species:
other: 3D human cornea tissue model
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Day of delivery: 14. Jul. 2015
Batch no.: 21562
Vehicle:
unchanged (no vehicle)
Controls:
other: negative and positive control vessels (in vitro study)
Amount / concentration applied:
The following amounts were applied to the tissues:

Tissue 1 51.5 mg
Tissue 2 49.4 mg
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Not applicable
Irritation parameter:
other: Relative absorbance (%)
Basis:
mean
Score:
107.5
Remarks on result:
other: Absorbance of test item compared to negative control

Findings and Results

Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:     

Table 1.Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate

1

2

3

4

5

6

7

8

Mean

Absorbance

0.035

0.038

0.035

0.035

0.034

0.036

0.036

0.036

0.036

 

The absorbance values of negative control, test item and positive control are given in the following table:

Table 2. Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation

Measurement

Negative Control

Positive Control

SABOSTAT A 300

Tissue 1 

1

1.289

0.453

1.440

2

1.368

0.457

1.438

Tissue 2 

1

1.358

0.456

1.446

2

1.360

0.456

1.447

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 1 (= corrected values).

Table3 Mean Absorbance Negative Control, Positive Control and Test Item

Designation

Negative Control

Positive Control

SABOSTAT A 300

Mean – blank (Tissue 1)

1.293

0.419

1.403

Mean – blank (Tissue 2)

1.323

0.420

1.411

Comparison of Formazan Production

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table 4 % Viability Positive Control and Test Item

Designation

Positive Control

SABOSTAT A 300

% Viability (Tissue 1)

32.0%

107.3%

% Viability (Tissue 2)

32.1%

107.8%

% Viability Mean

32.1%

107.5%

Assessment

Eye irritation is assessed using the criteria given in the following table (source: MatTek Corporation):

Table 5 Assessment of Eye Irritation

% Viability

Assessment

GHS classification

> 60 %

Non eye irritant

No GHS category
for eye irritation

60 %

Eye irritant

GHS category 1 or 2

 

 Validity

Validity criteria and results are stated in the following table:

Table 6 Validity

Criterion

Demanded

Found

OD of negative control

1.0 and≤ 2.6

1.3

% Formazan production of

positive control

< 60% of negative control

32.1%

Variation within replicates

< 20%

2.3% (negative control)
0.1% (positive control)
0.6% (test item)

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test system, the substance is considered as
not eye irritant in the EpiOcularTM Eye Irritation Test.
After treatment with the test item, the relative absorbance values increased to 107.5 %.
This value is well above the threshold for eye irritation potential (60%).
All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.3 (> 1.0 and < 2.6).
The positive control induced a decrease in the relative absorbance as compared to the negative control to 32.1% (< 60%). Variation within the replicates was acceptable (<20 %).
For these reasons, the result of the test is considered as valid.
Executive summary:

The substance was applied to a three-dimensional human cornea tissue model in duplicate for a 6 h exposure.

In average, 50.5 mg of the solid test item were applied to each tissue.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT which can be reduced to a blue formazan. Formazan production was measured by measuring the optical density (OD) of the resulting solution.

Deionised water was used as negative control, Methyl acetate was used as positive control.

The controls showed the expected results. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 ≤ mean OD ≤ 2.6, OD was 1.3. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 32.1 %. Variation within tissues was acceptable (< 20%).

 

After treatment with the test item, the relative absorbance values increased to 107.5 %.

This value is well above the threshold for eye irritation potential (60%).

 

Under the conditions of the test system, the substance is considered as

not eye irritant in the EpiOcularTMEye Irritation Test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three tissues of the human skin model EpiDermTMwere treated with the substance for 60 min.On average, 25 mg of the solid test item (wetted with 25 µL DPBS-buffer) were applied to each tissue and spread to match the tissue size (0.63 cm2; as indicated by supplier).DPBS-buffer was used as negative control, 5 % SDS solution was used as positive control.After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.8. The positive control showed clear irritating effects. Relative absorbance was reduced to 2.8 %.Variation within tissues was acceptable (< 18%). After the treatment with the test item, the relative absorbance values were increased to 119.6%. This value is well above the threshold for irritation potential (50%). Therefore,the substance is considered as not skin irritant in the Human Skin Model Test.

The substance was applied to a three-dimensional human cornea tissue model in duplicate for a 6 h exposure.

In average, 50.5 mg of the solid test item were applied to each tissue.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT which can be reduced to a blue formazan. Formazan production was measured by measuring the optical density (OD) of the resulting solution.

Deionised water was used as negative control, Methyl acetate was used as positive control.

The controls showed the expected results. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 ≤ mean OD ≤ 2.6, OD was 1.3. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 32.1 %. Variation within tissues was acceptable (< 20%).

 

After treatment with the test item, the relative absorbance values increased to 107.5 %.

This value is well above the threshold for eye irritation potential (60%).

 

Under the conditions of the test system, the substance is considered as

not eye irritant in the EpiOcularTMEye Irritation Test.

Justification for classification or non-classification

No evidence of adverse effects in 3 reliable (Klimisch 1) in vitro studies for skin irritation (OECD 439), for eye corrosion (BCOP, OECD 437) and eye irritation (EPIocular, OECD 405).

Thus the substance can be considered not irritant according to CLP regulation