Registration Dossier

Administrative data

Description of key information

Based on the results of the read across in vitro/in vivo studies, the test substance, 'Steardimonium hydroxypropyl hydrolysed wool', is considered to be irritating to both the skin and 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:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From July 28, 2017 to September 01, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
However, Guideline or SOP deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
not specified
Details on animal used as source of test system:
The reconstructed human epidermal model EpiDermTM (EPI-200-MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
Justification for test system used:
Initially the predictive capacity of the modified EpiDerm™ Skin Irritation Test (SIT) test method, using MatTek EpiDermTM tissue model EPI-200, underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test methods components of the original and updated ECVAM Performance Standards (PS). A modification of the original EpiDerm™ SIT was validated using the original ECVAM PS in 2008. In 2008, ESAC concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances.
Vehicle:
water
Details on test system:
Characterisation of the test system:

MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility. QC results for the specific lot of models received (Lot# 25839) were checked in-house for MatTek acceptance ranges with following outcome:
Morphology - PASS
Tissue viability - PASS
Skin barrier function (ET50 value for 1% Triton X-100) where ET50 is the time taken for 1% Triton X-100 to reduce the viability of the skin model to 50% relative to the negative control)- PASS
Sterility testing showed no contamination during long term antibiotic and antimycotic free culture- PASS
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg (nominal) test substance (62.8% active)
Duration of treatment / exposure:
60 ± 1 minute (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, ≥95% RH)
Duration of post-treatment incubation (if applicable):
42 ± 4h
Number of replicates:
Three tissue replicates per condition
Irritation / corrosion parameter:
% tissue viability
Remarks:
and c(Compared to the negative control)
Run / experiment:
Mean of 3 replicates
Value:
ca. 5.741
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Results

Prior to the study, the required compatibility checks confirmed that the test substance did not interfere with MTT. However, water colouration was observed. Therefore, additional viable tissues were run without MTT to control for colouration (Colourant Controls/No MTT). The background levels of interference from this control was subtracted from the initial viability derived from the main test to give an adjusted overall viability.

 

Table 1: Results summary

 

Percentage of viability

(relative to negative control)

Classification

Irritant (I)/non irritant (NI)

Test substance

5.741%

Irritant (I)

The test substance reduced the viability to below 50% and should be considered as Irritant to the skin.

Note that the final value was corrected after assessment of the colourant control.

 

Data Analysis:

 

Table 2: Viability measurements after 60 min (± 1min) of application and 46h (+9 mins) post-incubation of test and reference substances and controls

Condition

Tissue #

Raw data

Blank corrected data

Mean OD

% of Viability

Aliquot 1

Aliquot 2

Aliquot 1

Aliquot 2

NC

Tissue 1

1.911

1.865

1.737

1.691

1.714

105.0

Tissue 2

1.265

1.371

1.091

1.197

1.144

77.8

Tissue 3

1.856

1.595

1.682

1.421

1.552

95.0

PC

Tissue 1

0.265

0.282

0.091

0.108

0.100

6.1

Tissue 2

0.232

0.232

0.058

0.058

0.058

3.6

Tissue 3

0.231

0.291

0.057

0.117

0.087

5.3

TA3

Tissue 1

0.304

0.303

0.130

0.129

0.130

8.0

Tissue 2

0.385

0.376

0.211

0.202

0.207

12.7

Tissue 3

0.286

0.288

0.112

0.114

0.113

6.9

NC: negative control (DPBS), PC: Positive control (SDS 5%), TA3: Test substance. 

Note: Rounded figures used. NC tissue 2 was removed from further analysis as it was an outlier value as can be seen from the OD values and % viability.

 

Table 3: Mean and SD of cell viability measurements and of viability percentages after 60 min (± 1min) of application and 46h (+9 mins) post-incubation

Name

Code

Mean of OD

SD of OD

Mean of viability (%)

SD of viability (%)

CV %

Classification

DPBS

NC

1.633

0.115

100

7.036

7.036

Non-Irritant

SDS 5%

PC

0.082

0.021

5.011

1.304

26.016

Irritant

Test substance

TA3

0.150

0.050

9.185

3.056

33.270

Irritant

NC: Negative control (DPBS), PC: Positive control (SDS 5%), TA3: Test substance.

Prediction model of irritancy: test substances that reduce the viability to 50% or below are irritant (I), test substances with a percentage viability above 50% are considered to be non-irritant (NI).

Note: Rounded figures used.

Table 4: Mean % viabilities obtained from additional tests to correct for colourant interference

Test Substance

Colourant Controls (CC/No MTT): % viability

Initial % Viability

Corrected % Viability

TA3

3.444

9.185

5.741%

 TA3: Test substance.

Evaluation of the results

Results were checked against the following acceptance criteria: 

 

Description

Actual values

PASS/FAIL

Acceptance criterion 1

The mean OD570of the

negative control (treated with DPBS) tissues is≥ 0.8 and ≤ 2.8

1.633

PASS

Acceptance criterion 2

The mean of the positive control relative percentage viability must be ≤ 20% of the mean of the negative controls

5.011

PASS

Acceptance criterion 3

The standard deviation of OD values for triplicate skin models in each experimental condition must be < 18%

NC: 7.036

PC: 1.304

TA3: 3.056

PASS

Acceptance criterion 4

The mean OD of the 6 wells containing extraction solvent alone (blanks) should be ≤ 0.1

0.174

FAIL

All acceptance criteria were met with the exception of criterion 4:

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the XCellR8 laboratory and meet laboratory internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 historical data, based on blanks obtained during 66 historical runs), therefore this is not considered to be an issue in the interpretation of this study data. The study author concluded that this SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.

 

Interpretation of results following Prediction Model

 

1) A test substance is considered to be an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is ≤50%.

 2) A test substance may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is >50%.

The final percentage of viability obtained with the test substance test substance was 5.741%, therefore it was considered as Irritant to the skin.

Interpretation of results:
other: Category 2 (irritant) based on EU CLP criteria
Conclusions:
Based on the results of the read across study, the test substance is considered to be irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%) using Reconstructed Human Epidermis (RHE) test method, according to OECD Test Guideline 439, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% relative humidity (RH), after which 30 µL read across substance and reference substances (negative control: sterile Dulbecco’s phosphate buffered saline and positive control: sodium dodecyl sulphate (5% in water)) were applied topically for 60 ± 1 minute in triplicate, followed by rinsing steps and a 46 h + 9 mins post-dose incubation at 37°C, 5% CO2, ≥95% RH. Medium was changed on Day 2. MTT viability test was conducted and readings at 570 nm without reference filter were taken on Day 3. A test substance was considered to be an irritant to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. Based on the results, the final percentage of viability obtained with the read across substance was determined to be 5.741%; therefore it was considered as irritating to the skin. The Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. The study author concluded that this deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained. Hence, the study results qualified the acceptance criteria. Under the study conditions, the read across substance was determined to be irritating to the skin (XCellR8, 2017). Based on the results of the read across study, a similar irritation potential is expected for the test substance, 'Steardimonium hydroxypropyl hydrolysed wool'.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From July 28, 2017 to September 01, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
However, Guideline or SOP deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
not specified
Details on animal used as source of test system:
The reconstructed human epidermal model EpiDermTM (EPI-200-MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
Justification for test system used:
Initially the predictive capacity of the modified EpiDerm™ Skin Irritation Test (SIT) test method, using MatTek EpiDermTM tissue model EPI-200, underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test methods components of the original and updated ECVAM Performance Standards (PS). A modification of the original EpiDerm™ SIT was validated using the original ECVAM PS in 2008. In 2008, ESAC concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances.
Vehicle:
water
Details on test system:
Characterisation of the test system:

MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility. QC results for the specific lot of models received (Lot# 25839) were checked in-house for MatTek acceptance ranges with following outcomes:
Morphology - PASS
Tissue viability - PASS
Skin barrier function (ET50 value for 1% Triton X-100) where ET50 is the time taken for 1% Triton X-100 to reduce the viability of the skin model to 50% relative to the negative control)- PASS
Sterility testing showed no contamination during long term antibiotic and antimycotic free culture- PASS
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 µL of neat test substance (60.7% active)
Duration of treatment / exposure:
60 ± 1 minute (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, ≥95% RH)
Duration of post-treatment incubation (if applicable):
42 ± 4h
Number of replicates:
Three tissues replicates per condition
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of 3 replicates
Value:
ca. 3.179
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Results

Prior to the study, the required compatibility checks confirmed that the test substance interfered with MTT and water colouration was observed. Therefore, additional freeze killed (FK) controls were included to control for MTT interference and additional viable tissues were run without MTT to control for colouration (Colourant controls/No MTT). The background levels of interference from these controls were subtracted from the initial viability from the main test to give an adjusted overall viability.

Table 1: Results summary

Name

Mean of OD

SD of OD

Mean of viability (%)

SD %

Prediction

Negative control (DPBS)

1.633

0.115

100

7.036

Non Irritant

Positive control (SDS 5%)

0.082

0.021

5.011

1.304

Irritant

Test substance

0.100

0.043

3.179* (corrected)

2.634

Irritant

The test substance reduced the viability to below 50% and should be considered as Irritant to the skin. The final value was corrected after assessment of the colourant and freeze killed controls.

Data Analysis

Table 2 : Viability measurements after 60 min (± 1min) of application and 46h (+9 mins) post-incubation of test and reference substances and controls 

Condition

Tissue #

Raw data

Blank corrected data

Mean OD

% of Viability

Aliquot 1

Aliquot 2

Aliquot 1

Aliquot 2

NC

Tissue 1

1.911

1.865

1.737

1.691

1.714

105.0

Tissue 2

1.265

1.371

1.091

1.197

1.144

77.8

Tissue 3

1.856

1.595

1.682

1.421

1.552

95.0

PC

Tissue 1

0.265

0.282

0.091

0.108

0.100

6.1

Tissue 2

0.232

0.232

0.058

0.058

0.058

3.6

Tissue 3

0.231

0.291

0.057

0.117

0.087

5.3

TA2

Tissue 1

0.309

0.337

0.135

0.163

0.149

9.1

Tissue 2

0.24

0.257

0.066

0.083

0.075

4.6

Tissue 3

0.242

0.255

0.068

0.081

0.075

4.6

NC: negative control (DPBS), PC: Positive control (SDS 5%), TA2: Test substance. 

Note: Rounded figures used. NC tissue 2 was removed from further analysis as it was an outlier value as can be seen from the OD values and % viability.

Table 3: Mean and SD of cell viability measurements and of viability percentages after 60 min (± 1min) of application and 46h (+9 mins) post-incubation 

Name

Code

Mean of OD

SD of OD

Mean of viability (%)

SD of viability (%)

CV %

Classification

DPBS

NC

1.633

0.115

100.000

7.036

7.036

Non-Irritant

SDS 5%

PC

0.082

0.021

5.011

1.304

26.016

Irritant

Test substance

TA2

0.100

0.043

6.103

2.634

43.156

Irritant

NC: Negative control (DPBS), PC: Positive control (SDS 5%), TA2: Test substance.

Prediction model of irritancy: test substances that reduce the viability to 50% or below are irritant (I), test substances with a percentage viability above 50% are considered to be non-irritant (NI).

Note: Rounded figures used.

Table 4: Mean % viabilities obtained from additional tests to correct for MTT and colourant interference.

Test substance

MTT Interference Control (FK):

% viability

Colourant Controls (CC/No MTT): % viability

Initial % Viability

Corrected % Viability

TA2

1.347

1.577

6.103

3.179%

 TA2: Test substance.

Evaluation of the results

 Results were checked against the following acceptance criteria:

 

Description

Actual values

PASS/FAIL

Acceptance criterion 1

The mean OD570of the negative control (treated with DPBS) tissues is≥ 0.8 and ≤ 2.8

 

1.633

PASS

Acceptance criterion 2

The mean of the positive control relative percentage viability must be ≤ 20% of the mean of the negative controls.

 

5.011

PASS

Acceptance criterion 3

The standard deviation of OD values for triplicate skin models in each experimental condition must be < 18%

 

NC: 7.036

PC: 1.304

TA2: 2.634

PASS

Acceptance criterion 4

The mean OD of the 6 wells containing extraction solvent alone (blanks) should be ≤ 0.1.

 

0.174

FAIL

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the XCellR8 laboratory and meet our current internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 historical data, based on blanks obtained during 66 historical runs), therefore this is not considered to be an issue in the interpretation of this study data. This SOP and guideline deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained.

 

Interpretation of results following Prediction Model

1) A test substance is considered to be an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is ≤50%.

2) A test substance may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is >50%.

The final percentage of viability obtained with the test substance test substance was 3.179%, therefore it was considered as Irritant to the skin.

Interpretation of results:
other: Category 2 (irritant) based on EU CLP criteria
Conclusions:
Based on results of the read across study, the test substance is considered to be irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active: 60.7%), using Reconstructed Human Epidermis (RHE) test method, according to OECD Test Guideline 439, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% relative humidity (RH), after which 30 µL read across substance and reference substances (i.e., negative control: sterile Dulbecco’s phosphate buffered saline and positive control: sodium dodecyl sulphate (5% in water)) were applied topically for 60 ± 1 minute in triplicate, followed by rinsing steps and a 46 h + 9 mins post-dose incubation at 37°C, 5% CO2, ≥95% RH. Medium was changed on Day 2. MTT viability test was conducted and readings at 570 nm without reference filter were taken on Day 3. A test substance was considered to be an irritant to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. Based on the results, the final percentage of viability obtained with the read across substance was determined to be 3.179%; therefore it was considered as irritating to the skin. The Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. The study author concluded that this deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained. Hence, the study results qualified the acceptance criteria. Under the study conditions, the read across substance was determined to be irritating to the skin (XCellR8, 2017). Based on the results of the read across study, a similar irritation potential is expected for the test substance, 'Steardimonium hydroxypropyl hydrolysed wool'.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From January 24, 2018 to February 01, 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
The reconstructed human epidermal model EpidermTM (EPI-200 MatTek Corporation)
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Justification for test system used:
The EpiDermTM skin model and assay for skin corrosion testing is endorsed by OECD TG 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system
The reconstructed human epidermal model EpidermTM (EPI-200 MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differential model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Characterisation of the test system
MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility. QC results for the specific lot of models received (Lot# 25876) were checked in-house for MatTek acceptance ranges with the following outcome:
- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 1 % Triton X-100) where ET50 is the time taken for 1 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Single topical application of 15 μL sterile water plus neat test substance
50 μL reference substances
Duration of treatment / exposure:
3 and 60 minutes at 37°C, 5 % CO2, 95 % RH
Number of replicates:
3 replicates each for test substance, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
ca. 95.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
ca. 53.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive
Other effects / acceptance of results:
Prior to the assay, the test substance was checked for interference (water coloration or MTT interference) and found not to interfere.

Results

Table 1: Cell viability measurements after 3 minutes of application

Name

Tissue n°

3 min endpoint

Aliq. 1

Aliq. 2

mean

OD Mean

viability

Mean

SD

CV

[%]

[%]

[%]

[%]

NC

1

1.642

1.604

1.623

1.561

104.0

100.0

3.4

3.4

2

1.536

1.516

1.526

 

97.7

 

 

 

3

1.554

1.516

1.535

 

98.3

 

 

 

PC

1

0.374

0.356

0.365

0.379

23.4

24.3

0.8

3.3

2

0.388

0.382

0.385

 

24.6

 

 

 

3

0.390

0.385

0.387

 

24.8

 

 

 

TA2

1

1.496

1.459

1.477

1.486

94.6

95.2

0.6

0.6

2

1.509

1.483

1.496

 

95.8

 

 

 

3

1.478

1.491

1.484

 

95.1

 

 

 

NC: negative control (H2O), PC: Positive control (KOH 8N), TA2: Test substance

Table 2: Cell viability measurements after 1 h of application

Name

Tissue n°

1h endpoint

 

Aliq. 1

Aliq. 2

mean

OD Mean

viability

Mean

SD

CV

[%]

[%]

[%]

[%]

NC

1

1.565

1.551

1.558

1.548

100.7

100.0

3.9

3.9

2

1.492

1.473

1.483

 

95.8

 

 

 

3

1.617

1.586

1.602

 

103.5

 

 

 

PC

1

0.062

0.052

0.057

0.048

3.7

3.1

0.7

21.7

2

0.035

0.038

0.037

 

2.4

 

 

 

3

0.047

0.054

0.051

 

3.3

 

 

 

TA2

1

0.969

0.954

0.962

0.830

62.1

53.6*

12.1

22.5

2

 

 

 

 

 

 

 

 

3

0.692

0.702

0.697

 

45.1

 

 

 

*corrected value in (one outlier removed for TA2, i.e., second tissue readings)

NC: negative control (H2O), PC: Positive control (KOH 8N), TA2: Test substance

 

Table 3: Mean and SD of cell viability measurements after 3 minutes and 1 h application

 

3 min

1 h

Mean of viability [%]

SD of viability

CV(%)

Mean of viability [%]

SD of viability

CV(%)

NC

100.0

3.4

3.4

100.0

3.9

3.9

PC

24.3

0.8

3.3

3.1

0.7

21.7

TA2

95.2

0.6

0.6

53.6

12.1

22.5

NC: negative control (H2O), PC: Positive control (KOH 8N), TA2: Test substance.

 

Table 4: Results Summary

Test substance

Test substance ID

Viability after 3 minutes application

(% to negative control)

Viability ≥ 50% after 3 min (Yes/No)

Viability after 1h application

(% to negative control)

Viability ≥ 15% after 1h (Yes/No)

Corrosive (C)/Non corrosive(NC)

Test substance

TA2

95 .2%

Yes

53.6%

Yes

NC

The test substance did not reduce the viability below 50% after 3 min nor below 15% after 1h and should be considered as non-corrosive. Note that the final results are presented here (without outliers).

Acceptance criteria

 

 

Actual values

Pass/Failed

Acceptance criterion 1

The mean OD570of the negative control tissues must be ≥0.8.

 

3 min: 1.561

1h: 1.548

 

Pass

Acceptance criterion 2

The mean of the positive control relative percentage viability, after 1 hour exposuremust be < 15% of the mean of the negative control.

 

3.1%

Pass

Acceptance criterion 3

In the range between 20% and 100% viability, the coefficient of variation (CV) is an additional acceptance criterion.It should not exceed 0.3(i.e 30%).

 

CV values

3min

1h

NC

3.4

3.9

PC

3.3

21.7

TA2

0.6

22.5

Pass

Interpretation of results and skin corrosion Prediction Model

 

The cut-off values for the prediction of human skin corrosion are as follows:

 

Step 1

A test substance is classified "corrosive", if the relative tissue viability after3 mintreatment with a test material is decreased below 50%.

In addition, those materials classified "non-corrosive" after3 min(viability ≥ 50%) are classified "corrosive" if the relative tissue viability after1 htreatment with a test material is decreased below 15%.

 

Mean tissue viability(expressed as % of negative control)

Prediction

3 min < 50%

corrosive

3 min ≥ 50%and1 h: < 15%

corrosive

3 min ≥ 50%and1 h: ≥ 15%

non-corrosive

 

Step 2 (if test substance is classified as corrosive in step 1)

A test substance is classified "corrosive, optional Sub-Category 1A", if the relative tissue viability after3 mintreatment with a test material is decreased below 25%.

A test substance is classified "corrosive, optional Sub-Category 1B/1C", if the relative tissue viability after3 mintreatment with a test material is ≥25%.

 

Mean tissue viability(expressed as % of negative control)

Prediction

3 min < 25%

Corrosive,

optional Sub-category 1A

3 min ≥ 25%

Corrosive,

optional Sub-categories 1B and 1C

Conclusion for test substance

Test substance evaluated for skin corrosion following OECD guideline TG 431 and using EpiDermTM tissue model was non-corrosive.

Interpretation of results:
other: non-corrosive based on CLP criteria
Conclusions:
Based on results of the read across study, the test substance is considered to be non-corrosive to the skin.


Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (51% active), using Reconstructed Human Epidermis (RHE) cells, according to OECD Guideline 431, in compliance with GLP. EpiDermTM tissues were kept overnight at 4°C. On Day 1, the tissues were pre-incubated for 1 h at 37°C, 5% CO2, 95% RH. After incubation, tissues were exposure to test (15 μL water plus neat read across substance) and reference substances (50 μL sterile water as negative control and 50 μL Potassium hydroxide as positive control) in triplicates for 3 and 60 minutes. After 3 minutes and 1 h treatment, the read across substance and the reference substances were rinsed off from the tissues. Cell viability of the tissues was evaluated by addition of MTT on Day 2 and final MTT assay testing and measurements were performed. Results were compared to negative control. All validity criteria for the performed test were met. After 3 minutes and 1 h treatment, the mean viability values obtained with the read across substance were determined to be 95.2% and 53.6%, respectively, which is well above the corrosive limits of 50 and 15% respectively (XCellR8, 2018). Based on results of the read across study, similar absence of corrosive potential can be expected for the test substance, 'Steardimonium hydroxypropyl hydrolysed wool'.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From September 20, 2017 to September 22, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
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:
yes
Remarks:
Deviation was considered to have not affected the integrity or validity of the study.
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
Deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
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 refrigerated on arrival and used within 24 h of receipt.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL test (undiluted) or reference substance
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
32 ± 1ºC for 120 minutes
Number of animals or in vitro replicates:
Triplicates

Details on study design:
Preparation of corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. 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 ± 1ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of corneas and opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.

Treatment of corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance or control substances were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number HN60FW and PB95SY.

Application of sodium fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Data evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity measurement
The change in opacity for each cornea (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.

Permeability measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test substance induced a response through only one of the two endpoints.

Visual observation
The condition of the cornea was visually assessed post treatment.

Major Computerized Systems
The following computerized systems were used in the study:
• Delta Building Monitoring System.
• Labtech LT-4500 microplate reader and LT-com software
Irritation parameter:
in vitro irritation score
Value:
ca. 32.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction could be made about eye irritation potential
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied. The study has met validity criteria.

Results

Individual and mean corneal opacity and permeability measurement


Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control*

1

3

3

3

0

 

0.000

 

 

2

5

6

6

1

 

0.002

 

 

3

3

3

4

1

 

0.001

 

 

Mean

 

 

 

0.7

 

0.001

 

0.7

Positive Control*

4

4

36

34

30

29.3

0.664

0.663

 

5

4

35

35

31

30.3

1.280

1.279

 

6

2

35

35

33

32.3

1.067

1.066

 

Mean

 

 

 

 

30.7

 

1.003

45.7

Test substance

7

4

14

20

16

15.3

1.241

1.240

 

8

4

20

20

16

15.3

0.745

0.744

 

9

3

12

15

12

11.3

1.765

1.764

 

Mean

 

 

 

 

14.0

 

1.249

32.7

OD= Optical density            

* = Control data shared with Envigo - Shardlow study number HN60FW and PB95SY

Corneal epithelium condition post treatment

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

1

Clear

Clear

2

Clear

Clear

3

Clear

Clear

Positive Control

4

Cloudy

Cloudy

5

Cloudy

Cloudy

6

Cloudy

Cloudy

Test substance

7

Partly Cloudy

Cloudy

8

Partly Cloudy

Cloudy

9

Partly Cloudy

Cloudy

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

 

Treatment

In Vitro Irritancy Score

Test substance

32.7

Negative Control

0.7

Positive Control

45.7

 

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:
other: inconclusive results based on CLP criteria
Conclusions:
Based on the results of the read across study, no clear prediction of eye irritation can be made for the test substance; although the IVIS scores are indicative of absence of a corrosive potential.



Executive summary:

An in vitro study was conducted to determine the eye corrosion potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%), using the Bovine corneal Opacity Test (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Appropriately prepared bovine cornea, in triplicates were exposed to 0.75 mL of the read across substance (undiluted) and reference substances (Negative control: sodium chloride 0.9% w/v, Positive control: Ethanol). The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes. At the end of the exposure period the read across substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’s Minimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number HN60FW and PB95SY. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. 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 for read across substance, negative control and positive control were reported to be 32.7, 0.7 and 45.7 respectively. The study had met all validity criteria, except for negative control, however, the deviation was considered to have not affected the integrity or validity of the study. Under the study conditions, based on the IVIS score of the read across substance, which is >3 and <55%, no prediction of eye irritation potential could be made (Envigo, 2018). Based on the results of the read across study, similar eye corrosion potential is expected for the test substance, 'Steardimonium hydroxypropyl hydrolysed wool'.

Endpoint:
eye irritation: in vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From March 31, 1981 to April 14, 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
The substance to be tested is applied in a single dose to one of the eyes of the experimental animal; the untreated eye serves as the control. The degree of eye irritation/corrosion is evaluated by scoring lesions of conjunctiva, cornea, and iris, at specific intervals. Other effects in the eye and adverse systemic effects are also described to provide a complete evaluation of the effects. The duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
24 h
Observation period (in vivo):
24, 48, and 72 h
Number of animals or in vitro replicates:
6 rabbits
Details on study design:
Procedure
This test was designed to determine the ocular irritation potential of the test substance in rabbits. The procedure followed was a modification of that described by J.H. Draize. Six (6) New Zealand White rabbits, weighing approximately 2 kg and about 3 months of age, Sex unspecified, were obtained from a suitably licensed dealer. Animals were checked carefully upon receipt for ocular defects, diarrhea and dehydration, respiratory difficulties, postural deficiencies and general condition. Animals were acclimated at least 3 d prior to test initiation. They were housed in galvanized or stainless steel cages and identified through individual markings on the outer ear of each animal, as well as a cage label. Diet consisted of a growth and maintenance ration from a commercial producer, as well as water, libitum. Immediately prior to test initiation, the animals were placed in wooden restrainers. A dose of 0.1 mL of the test substance was placed on one eye of each animal by gently pulling the lower lid away from the eyeball to form a cup into which the test substance was dropped. The eyelids were gently held together for 1 second. The contralateral eye, remaining untreated, served as control. If any of the test substance remained in the eye at 24 h. the eye was washed out with lukewarm water after the 24 h reading. Observations of ocular irritation were recorded 24, 48 and 72 h following instillation of the test substance. Additional readings were made at 4 and 7 d after application if irritation persisted. An animal was considered as exhibiting a positive reaction if the test substance produced any of the following: ulceration of the cornea other than a fine stippling, opacity of the cornea other than a slight dulling of the normal luster, inflammation of the iris other than a slight deepening of the folds or slight circumcorneal injection of the blood vessels, obvious conjunctival swelling with partial eversion of the lids or a diffuse crimson-red with individual vessels not discernible. If two (2) or more animals exhibited a positive reaction, the test substance was considered an ocular irritant (Unless the test was repeated with another six animals without positive reactions).
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Reversibility:
other: No adverse effect reported
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
2
Reversibility:
other: No adverse effected reported
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Reversibility:
other: No swelling observed
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
20
Reversibility:
other: No adverse effects observed
Remarks on result:
no indication of irritation

Summary of eye irritation

Rabbit

Day

Cornea (A*B*5) +

Iris (A*5) +

Conjunctivae (A+B+C)*2 -

 

 

Total score*

Opacity (A)

Area of cornea involved (B)

Values (A)

Redness (A)

Chemosis (B)

Discharge ©

1

1

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

 

4

-

-

-

-

-

-

-

 

7

-

-

-

-

-

-

-

2

1

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

 

4

-

-

-

-

-

-

-

 

7

-

-

-

-

-

-

-

3

1

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

 

4

-

-

-

-

-

-

-

 

7

-

-

-

-

-

-

-

4

1

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

 

4

-

-

-

-

-

-

-

 

7

-

-

-

-

-

-

-

5

1

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

 

4

-

-

-

-

-

-

-

 

7

-

-

-

-

-

-

-

6

1

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

 

4

-

-

-

-

-

-

-

 

7

-

-

-

-

-

-

-

Average

1

 

 

 

 

 

 

0

 

2

 

 

 

 

 

 

0

 

3

 

 

 

 

 

 

0

 

4

 

 

 

 

 

 

-

 

7

 

 

 

 

 

 

-

*Total score possible/animal/observation interval = 110

Interpretation of results:
study cannot be used for classification
Remarks:
no concentration specified
Conclusions:
Based on the results of the read across study, the test substance is considered to be non-irritating to the eyes.
Executive summary:

A study was conducted to determine the eye irritation potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active: not specified) according to the Draize method, in compliance with GLP. Six (6) New Zealand white rabbits, free from visible ocular defects, each received a single intraocular application of 0.1 mL of the test substance (concentration not specified) in one eye. The contralateral eye, remaining untreated, served as a control. The test substance was used as received (pH - 6.3). The eyes of all animals remained unwashed for 24 h. Observations of corneal opacity, iritis, and conjunctivitis were recorded 24, 48 and 72 h after treatment, and at 4 and 7 d, if irritation persisted. No irritation effects were observed in all animals. The overall, Draize score was determined to 0. Under the study conditions, the test substance was determined to be non-irritating to the eyes (CPT, 1981). Based on the results of the read across study, similar eye irriation response is expected for the test substance, 'Steardimonium hydroxypropyl hydrolysed wool'.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From September 20, 2017 to September 22, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
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:
yes
Remarks:
Deviation was considered to have not affected the integrity or validity of the study.
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
Deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
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 refrigerated on arrival and used within 24 h of receipt.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL test (undiluted) or reference substance
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
32 ± 1ºC for 120 minutes
Number of animals or in vitro replicates:
Triplicates
Details on study design:
Preparation of corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. 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 ± 1ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of corneas and opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.

Treatment of corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance or control substances were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number PQ52DX and PB95SY.

Application of sodium fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Data evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity measurement
The change in opacity for each cornea (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.

Permeability measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test substance induced a response through only one of the two endpoints.

Visual observation
The condition of the cornea was visually assessed post treatment.

Major Computerized Systems
The following computerized systems were used in the study:
• Delta Building Monitoring System.
• Labtech LT-4500 microplate reader and LT-com software
Irritation parameter:
in vitro irritation score
Value:
ca. 6.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction could be made about eye irritation potential

Results

Individual and mean corneal opacity and permeability measurement

Treatment

Cornea Number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post Incubation

Post-Incubation - Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control*

1

3

3

3

0

 

0.000

 

 

2

5

6

6

1

 

0.002

 

 

3

3

3

4

1

 

0.001

 

 

 Mean

 

 

 

0.7

 

0.001

 

0.7

Positive Control*

4

4

36

34

30

29.3

0.659

0.658

 

5

4

35

35

31

30.3

1.276

1.275

 

6

2

35

35

33

32.3

1.061

1.060

 

 Mean

 

 

 

 

30.7

 

0.998

45.6

Test Substance

13

1

8

5

4

3.3

0.328

0.327

 

14

4

13

8

4

3.3

0.225

0.224

 

15

3

13

7

4

3.3

0.151

0.150

 

 Mean

 

 

 

 

3.3

 

0.233

6.8

OD= Optical density            

* = Control data shared with Envigo - Shardlow study number PQ52DX and PB95SY

Corneal epithelium condition post treatment

Treatment

Cornea Number

Observation

Post Treatment

Post Incubation

Negative Control

1

Clear

Clear

2

Clear

Clear

3

Clear

Clear

Positive Control

4

Cloudy

Cloudy

5

Cloudy

Cloudy

6

Cloudy

Cloudy

Test Substance

13

Partly Cloudy

Slightly Cloudy

14

Partly Cloudy

Slightly Cloudy

15

Partly Cloudy

Slightly Cloudy

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

 

Treatment

In Vitro Irritancy Score

Test substance

6.8

Negative Control

0.7

Positive Control

45.6

 

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:
other: inconclusive results based on CLP criteria
Conclusions:
Based on the results of the read across study, no clear prediction of eye irritation can be made for the test substance; although the IVIS scores are indicative of absence of a corrosive potential.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), using the Bovine corneal Opacity Test (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Appropriately prepared bovine cornea, in triplicates were exposed to 0.75 mL of the read across substance (undiluted) and reference substances (Negative control: sodium chloride 0.9% w/v, Positive control: Ethanol). The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes. At the end of the exposure period the read across substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’sMinimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number PQ52DX and PB95SY. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. 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 for read across substance, negative control and positive control were reported to be 6.8, 0.7 and 45.6 respectively. The study had met all validity criteria, except for negative control, however, the deviation was considered to have not affected the integrity or validity of the study. Under the study conditions, based on the IVIS score of the read across substance, which is >3 and <55%, no prediction of eye irritation potential could be made (Envigo, 2018). Based on the results of the read across study, no clear prediction of eye irritation can be made for the test substance, 'Steardimonium hydroxypropyl hydrolysed wool'; although the IVIS scores are indicative of absence of a corrosive potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

Study 1:

An in vitro study was conducted to determine the skin corrosion potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), using Reconstructed Human Epidermis (RHE) cells, according to OECD Guideline 431, in compliance with GLP. EpiDermTM tissues were kept overnight at 4°C. On Day 1, the tissues were pre-incubated for 1 h at 37°C, 5% CO2, 95% RH. After incubation, tissues were exposure to read across (15 μL water plus neat read across substance) and reference substances (50 μL sterile water as negative control and 50 μL Potassium hydroxide as positive control) in triplicates for 3 and 60 minutes. After 3 minutes and 1 h treatment, the read across substance and the reference substances were rinsed off from the tissues. Cell viability of the tissues was evaluated by addition of MTT on Day 2 and final MTT assay testing and measurements were performed. Results were compared to negative control. All validity criteria for the performed test were met. After 3 minutes and 1 h treatment, the mean viability values obtained with the read across substance were determined to be 95.2% and 53.6%, respectively, which is well above the corrosive limits of 50 and 15% respectively. Under the study conditions, the read across substance was determined to be non-corrosive to the skin (XCellR8, 2018).

Study 2:

An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%) using Reconstructed Human Epidermis (RHE) test method, according to OECD Test Guideline 439, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% relative humidity (RH), after which 30 µL read across substance and reference substances (negative control: sterile Dulbecco’s phosphate buffered saline and positive control: sodium dodecyl sulphate (5% in water)) were applied topically for 60 ± 1 minute in triplicate, followed by rinsing steps and a 46 h + 9 mins post-dose incubation at 37°C, 5% CO2, ≥95% RH. Medium was changed on Day 2. MTT viability test was conducted and readings at 570 nm without reference filter were taken on Day 3. A test substance was considered to be an irritant to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. Based on the results, the final percentage of viability obtained with the read across substance was determined to be 5.741%; therefore it was considered as irritating to the skin. The Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. The study author concluded that this deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained. Hence, the study results qualified the acceptance criteria. Under the study conditions, the read across substance was determined to be irritating to the skin (XCellR8, 2017).

Study 3:

An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active: 60.7%), using Reconstructed Human Epidermis (RHE) test method, according to OECD Test Guideline 439, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% relative humidity (RH), after which 30 µL read across substance and reference substances (i.e., negative control: sterile Dulbecco’s phosphate buffered saline and positive control: sodium dodecyl sulphate (5% in water)) were applied topically for 60 ± 1 minute in triplicate, followed by rinsing steps and a 46 h + 9 mins post-dose incubation at 37°C, 5% CO2, ≥95% RH. Medium was changed on Day 2. MTT viability test was conducted and readings at 570 nm without reference filter were taken on Day 3. A test substance was considered to be an irritant to skin if the skin model viability after exposure and post-treatment incubation is ≤50%. Based on the results, the final percentage of viability obtained with the read across substance was determined to be 3.179%; therefore it was considered as irritating to the skin. The Optical Density (OD) values obtained with blanks were higher than 0.1 (0.174) causing a deviation from Acceptance Criteria 4. The study author concluded that this deviation was not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained. Hence, the study results qualified the acceptance criteria. Under the study conditions, the read across substance was determined to be irritating to the skin (XCellR8, 2017).

Based on results from the test substance and read across studies, the test substance, ‘Steardimonium hydroxypropyl hydrolysed wool' is considered to be irritating to the skin.

Eye:

Study 1:

An in vitro study was conducted to determine the eye irritation potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk' (active: 51%), using the Bovine corneal Opacity Test (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Appropriately prepared bovine cornea, in triplicates were exposed to 0.75 mL of the read across substance (undiluted) and reference substances (Negative control: sodium chloride 0.9% w/v, Positive control: Ethanol). The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes. At the end of the exposure period the read across substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’sMinimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number PQ52DX and PB95SY. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. 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 for read across substance, negative control and positive control were reported to be 6.8, 0.7 and 45.6 respectively. The study had met all validity criteria, except for negative control, however, the deviation was considered to have not affected the integrity or validity of the study. Under the study conditions, based on the IVIS score of the read across substance, which is >3 and <55%, no prediction of eye irritation potential could be made (Envigo, 2018).

Study 2:

An in vitro study was conducted to determine the eye corrosion potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%), using the Bovine corneal Opacity Test (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Appropriately prepared bovine cornea, in triplicates were exposed to 0.75 mL of the read across substance (undiluted) and reference substances (Negative control: sodium chloride 0.9% w/v, Positive control: Ethanol). The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes. At the end of the exposure period the read across substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’s Minimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed. The negative and positive control data was shared with Envigo study number HN60FW and PB95SY. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. 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 for read across substance, negative control and positive control were reported to be 32.7, 0.7 and 45.7 respectively. The study had met all validity criteria, except for negative control, however, the deviation was considered to have not affected the integrity or validity of the study. Under the study conditions, based on the IVIS score of the read across substance, which is >3 and <55%, no prediction of eye irritation potential could be made (Envigo, 2018).

Study 3:

A study was conducted to determine the eye irritation potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active: not specified) according to the Draize method, in compliance with GLP. Six (6) New Zealand white rabbits, free from visible ocular defects, each received a single intraocular application of 0.1 mL of the test substance (concentration not specified) in one eye. The contralateral eye, remaining untreated, served as a control. The test substance was used as received (pH - 6.3). The eyes of all animals remained unwashed for 24 h. Observations of corneal opacity, iritis, and conjunctivitis were recorded 24, 48 and 72 h after treatment, and at 4 and 7 d, if irritation persisted. No irritation effects were observed in all animals. The overall, Draize score was determined to 0. Under the study conditions, the test substance was determined to be non-irritating to the eyes (CPT, 1981).

Based on the in vitro BCOP studies with the read across substances, no clear conclusion could be drawn as per the Guideline. However, given the IVIS scores of the read across substance (i.e., 6.8 or 32.7), which is in more closer to the non-corrosive limit (i.e., <=3) or in between (>3 and <55%), together with positive skin irritation potential (which was assessed based on the in vitro skin irritation (RHE) study with the read across substances), indicate that a corrosive potential can be ruled out and the test substance, ‘Steardimonium hydroxypropyl hydrolysed wool’ can be considered to be more likely to be irritating to the eyes (in a worst case).)

Justification for classification or non-classification

Skin

Based on the results of read across in vitro skin corrosion/irritation studies, the test substance, ‘Steardimonium hydroxypropyl hydrolysed wool’, is concluded to warrant ‘Skin Irrit. 2; H315 - Causes skin irritation’ classification according to the EU CLP criteria (Regulation 1272/2008/EC).

 

Eye

Based on the available weight of evidence from read across in vitro or in vivo eye corrsosion/irriation studies together with in vitro skin corrosion/irritation studies, the test substance, ‘Steardimonium hydroxypropyl hydrolysed wool', is concluded to warrant ‘Eye Irrit. 2: H319 causes serious eye irritation’ classification according to the EU CLP criteria (Regulation 1272/2008/EC).