Registration Dossier

Administrative data

Description of key information

Based on the results of the in vitro studies on the test substance as well as read across substances, the test substance, mono- and di- C18-unsatd PSE + mono- and di- C18-unsatd PSE EO-5, is considered to be irritating to skin and corrosive to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
other: MatTek Corporation EpiDermTM skin model in vitro toxicity testing system
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
not applicable
GLP compliance:
yes
Test system:
human skin model
Remarks:
artificial three-dimensional model of human skin
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Validated, accurate and reliable method for the prediction skin irritationg and no-label (no-skin irritating) test substances.
Vehicle:
unchanged (no vehicle)
Details on test system:
Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
- 25 mg neat test substance
- 50 µL of water
- 8N potassium hydroxide
Duration of treatment / exposure:
3 minutes or 1 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
compared to negative control
Run / experiment:
3 min exposure
Value:
ca. 101
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
28% viability
Remarks on result:
other: no prediction of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
compared to negative control
Run / experiment:
1 h exposure
Value:
ca. 99
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
6% viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test substance did not reduce the viability to 50% or below and should be considered as non-corrosive to the skin.
Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance tested was determined to be non-corrosive to the skin.
Executive summary:

A study was conducted to determine the in vitro skin corrosion potential of the test substance, 'mono- and di- C16 -18 PSE and C16-18 AE20 PSE' using reconstructed human epidermis (RHE), according to a method similar to OECD Guideline 431, in compliance with GLP. Three replicates of 25 µg of the test substance (with 25 µL of distilled water), 50 µL of the negative control (water) and 8N positive control substance (8N Potassium hydroxide) were added to millicells MatTek Epiderm tissue samples for 3 minutes and 1 h time period. After exposure periods, the tissues were rinsed with phosphate buffered saline (PBS) to remove any residual material. After all tisses had been rinsed and dosed, they were placed for MTT assay. The MTT plates were then incubated for 3 h (at 37 ºC, 5% C02). After incubation period, the optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. The mean viability of cells exposed to the test substance were 101% and 99% of the negative control after 3 min and 1 h, respectively. The test substance did not reduce the viability to 50% or below and should be considered as non-corrosive to the skin. Under the study conditions, the test substance tested was determined to be non-corrosive to the skin (CPT, 2003).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
From May 16, 2017 to June 15, 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 read-across 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:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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# 25819) 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 25 μL sterile water and nominal 25 mg of test substance.
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:
103.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
93.8
Negative controls validity:
valid
Positive controls validity:
valid
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

1.768

1.795

1.781

1.749

101.9

100.0

2.5

2.5

2

1.697

1.701

1.699

 

97.2

 

 

 

3

1.761

1.771

1.766

 

101.0

 

 

 

TA3

1

1.784

1.802

1.793

1.814

102.5

103.7

2.4

2.3

2

1.871

1.855

1.863

 

106.5

 

 

 

3

1.767

1.805

1.786

 

102.1

 

 

 

PC

1

0.231

0.282

0.256

0.317

14.7

18.1

4.1

22.7

2

0.393

0.401

0.397

 

22.7

 

 

 

3

0.295

0.303

0.299

 

17.1

 

 

 

NC: negative control (H2O), PC: Positive control (KOH 8N), TA3: 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

1.842

1.900

1.871

1.777

105.3

100.0

5.0

5.0

2

1.656

1.729

1.692

 

95.2

 

 

 

3

1.733

1.806

1.769

 

99.5

 

 

 

TA3

1

1.481

1.485

1.483

1.668

83.4

93.8

10.1

10.7

2

1.653

1.710

1.681

 

94.6

 

 

 

3

1.805

1.876

1.840

 

103.5

 

 

 

PC

1

0.283

0.332

0.307

0.248

17.3

14.0

3.3

23.3

2

0.244

0.249

0.246

 

13.8

 

 

 

3

0.181

0.203

0.192

 

10.8

 

 

 

NC: negative control (H2O), PC: Positive control (KOH 8N), TA3: Test substance.

 

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

 

3min

1h

Mean of viability [%]

SD of viability

CV(%)

Mean of viability [%]

SD of viability

CV(%)

NC

100.0

2.5

2.5

100.0

5.0

5.0

TA3

103.7

2.4

2.3

93.8

10.1

10.7

PC

18.1

4.1

22.7

14.0

3.3

23.3

NC: negative control (H2O), PC: Positive control (KOH 8N), TA3: 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

TA3

103.7%

Yes

93.8%

Yes

NC

The test substance did not reduce the viability below 50% after 3 min nor below 15% after 1 h and should be considered as non-corrosive.

Acceptance criteria

 

 

Actual values

Pass/Failed

Acceptance criterion 1

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

 

1.749 after 3 min, 1.777 after 1h

Pass

Acceptance criterion 2

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

 

14.0%

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%).

 

NC: 2.5% after 3 min, 5.0% after 1h

PC: 22.7% after 3 min, 23.3% after 1h

TA3: 2.3% after 3 min, 10.7% after 1h

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:
Conclusions:
Based on the 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, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using Reconstructed Human Epidermis (RHE) cells, according to OECD 431 Guideline, 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 (25 mg) and reference substances (25 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 test substance and the reference substances were rinsed off from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT on Day 2. On Day 3, 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 substance were determined to be 103.7% and 93.8%, 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, 2017). Based on the results of the read across study, the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE', is considered to be non-corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 18, 2017 to June 23, 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 read-across 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:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
not considered to have affected the integrity or interpretation of the results as no equivocal results were obtained. For details please refer to 'any other information on methods and results incl. tables'
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
MatTek EpiDermTM tissue model EPI-200
Source species:
human
Cell type:
other: Normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differentiated model of the human epidermis.
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:
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 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.

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# 25819) 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:
After pre-wetting tissues with 25 µL DPBS, single topical application of nominal 25 mg neat test substance
Duration of treatment / exposure:
60 minutes of treatment
Duration of post-treatment incubation (if applicable):
42 ± 2 h
Number of replicates:
3 replicates for the test substance, positive and negative control
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
ca. 80.6
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Prior to the study, the required compatibility checks (as per SOP L0029) confirmed that the test substance did not interfere with MTT and no water colouration was observed.
- The test substance did reduce the viability below 50 % and should be considered as irritant to the skin.

All acceptance criteria were met with the exception of 1 criterion:

- The mean OD570 of the negative control (treated with DPBS) tissues is ≥0.8 and ≤2.8.
Result: 1.846
- The mean of the positive control relative percentage viability must be ≤20 % of the mean of the negative controls.
Result: 3.1 %
- The standard deviation of OD values for triplicate skin models in each experimental condition must be <18 %.
Results:
NC: 7.12 %
PC: 0.61 %
Test substance: 4.85 %

- The mean OD of the 6 wells containing extraction solvent alone (blanks) should be ≤0.1.
Result: 0.1673

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.1673) causing a deviation from acceptance criteria. 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 and meet current internal acceptance criteria of blank OD values <0.194, 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.




Results

Table 1: Viability measurements after 60 ±1 min of application and 42 ± 4 h 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

1.967

2.084

1.800

1.917

1.858

100.6

Tissue 2

2.095

2.182

1.928

2.015

1.971

106.8

Tissue 3

1.856

1.897

1.689

1.730

1.709

92.6

PC

Tissue 1

0.225

0.2

0.058

0.033

0.045

2.4

Tissue 2

0.237

0.217

0.070

0.050

0.060

3.2

Tissue 3

0.24

0.229

0.073

0.062

0.067

3.6

TA3

Tissue 1

1.65

1.865

1.483

1.698

1.590

86.1

Tissue 2

1.642

1.599

1.475

1.432

1.453

78.7

Tissue 3

1.581

1.597

1.414

1.430

1.422

77.0

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

Note: Rounded figures used.

 

Table 2: Mean and SD of cell viability measurements and of viability percentages after a 60 ±1 minute application and 42 ± 4 h post-incubation. 

Name

Code

Mean of OD

SD of OD

Mean of viability (%)

SD of viability (%)

CV %

Classification

DPBS

NC

1.846

0.131

100.0

7.12

7.12

Non-Irritant

SDS 5%

PC

0.057

0.011

3.1

0.61

19.51

Irritant

Test substance

TA3

1.488

0.090

80.6

4.85

6.02

Non-Irritant

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

Note: Rounded figures used.

Evaluation of the results

 

Results were checked against the following acceptance criteria:

 

Description

Actual values

PASS/FAIL

Acceptance criterion 1

The mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8

 

1.846

PASS

Acceptance criterion 2

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

 

3.1%

PASS

Acceptance criterion 3

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

  

NC: 7.12%

PC: 0.61%

TA3: 4.85%

PASS

Acceptance criterion 4

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

 

0.1673

FAIL*

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

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.1673) 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 XCellR8 historical data, based on blanks obtained during the last 66 studies), 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 percentage of viability obtained with the test substance was 80.6%, therefore it is considered as Non-Irritant to the skin.

Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Based on the results of the read across study, the test substance is considered to be non-irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE' using Reconstructed Human Epidermis (RHE) cells, according to OECD 439 Guideline, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, 95% RH. On Day 1, the tissues in triplicate were exposed to nominal 25 mg of test substance and 30 µL reference substances, applied topically for 60 ±1 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, 95% RH), followed by rinsing steps and a 42 ± 4 h post-dose incubation at 37°C, 5% CO2, 95%RH). On Day 2, the medium was changed and on Day 3, MTT viability test with readings at 570 nm without reference filter was performed. 30 µL of DPBS and 5% SDS were used as negative control and positive control, respectively. Viability of the tissues was assessed in MTT test and compared to the negative control. The percentage of viability obtained with the substance was 80.6%, which is well above the irritant limit of 50%. The study met all the validity criteria. Under the study conditions, the read across substance was determined be non-irritant to skin (XCellR8, 2017). Based on the results of the read across study, the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE' is considered to be non-irritating to the skin.

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:
experimental study
Adequacy of study:
key study
Study period:
July 04, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
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. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the 20% w/v test substance solution in sodium chloride 0.9% w/v or control substances (sodium chloride 0.9% w/v as negative control, 20% w/v imidazole solution in sodium chloride 0.9% w/v as positive control)
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
32 ± 1ºC for 90 minutes
Number of animals or in vitro replicates:
Three replicates per substance
Irritation parameter:
in vitro irritation score
Run / experiment:
Test substance
Value:
ca. 89.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Remarks:
The deviation was considered to have not affected the integrity or validity of the study
Remarks on result:
positive indication of irritation
Remarks:
Category 1 (irreversible effects on the eye) based on EU CLP criteria
Other effects / acceptance of results:
The positive control group had an overall IVIS of 127.0, which was marginally higher than the criteria range set for an acceptable test. However, as the score was only marginally exceeded, it was decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant. This deviation was considered to have not affected the integrity or validity of the study. The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

Results

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in below table:

Table1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment

Cornea Number

Opacity

Permeability Optical Density (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control #

1

3

4

1

 

0.012

 

 

2

3

4

1

 

0.000

 

 

3

2

4

2

 

0.000

 

 

Mean

 

 

1.3

 

0.004

 

1.4

Positive
Control #

4

2

105

103

101.7

3.965

3.961

 

5

2

88

86

84.7

1.895

1.891

 

6

2

83

81

79.7

1.820

1.816

 

Mean

 

 

 

88.7

 

2.556

127.0

Test Substance

7

3

94

91

89.7

0.504

0.500

 

8

2

85

83

81.7

0.402

0.398

 

9

2

79

77

75.7

0.468

0.464

 

Mean

 

 

 

82.3

 

0.454

89.1

#= Control data shared with Envigo - Shardlow study number LM55TK and XL29CC

 

Corneal Epithelium Condition

The condition of each cornea is given in below table:

Table 2: Corneal Epithelium Condition Post Treatment

Treatment

Cornea Number

Observation
Post Treatment

Negative Control #

1

Clear

2

Clear

3

Clear

Positive Control #

4

Cloudy

5

Cloudy

6

Cloudy

Test Substance

7

Cloudy

8

Cloudy

9

Cloudy

#= Control data shared with Envigo - Shardlow study number LM55TK and XL29CC


 The corneas treated with the test substance were cloudy post treatment. The corneas treated with the negative control substance were clear post treatment. The corneas treated with the positive control substance were cloudy post treatment.

 

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Substance

89.1

Negative Control

1.4

Positive Control

127.0

 

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was above the range of 65.1 to 123.3. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation. The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

 

Conclusion

Based on the study results, the test substance was classified as Category 1 (irreversible effects on the eye) based on GHS criteria

Interpretation of results:
other: Category 1 (irreversible effects on the eye) based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance was determined as inducing serious eye damage and classified as Category 1 (irreversible effects on the eye) based on EU CLP criteria.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the the test substance, ‘mono- and di- C16-18 PSE and C16-18 AE20 PSE’, using the Bovine Corneal Opacity and Permeability (BCOP) method, according to the OECD Guideline 437 and EU Method B.47, in compliance with GLP. The test substance was applied to test system at a concentration of 20% w/v in 0.9% w/v sodium chloride for 240 minutes followed by post exposure period at 32 ± 1ºC for 90 minutes. Negative and positive control substances were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The test substance IVIS was determined to be 89.1, which is well above the corrosive limit of 55. Therefore, the test substance was classified as Category 1 (irreversible effects on the eye) based on GHS/EU CLP criteria. The positive control IVIS was 127, which was outside the range of 65.1 to 123.3, however, as the score was only marginally exceeded, study author decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant. Therefore, this deviation was considered to have not affected the integrity or validity of the study. The negative control gave opacity of ≤1.3 and permeability ≤0.004, therefore the negative control acceptance criteria were satisfied. The test was considered to pass all the validity criterias. Under study conditions, the test substance was considered to be corrosive and classified as Eye Damage 1 (causes serious eye damage) based on EU CLP criteria (Envigo, 2017).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
other: HET-CAM test
Version / remarks:
modification of that described by Kemper and Luepke
GLP compliance:
yes
Species:
other: Chorioallantoic membrane (CAM) of chick embryo
Details on test animals or tissues and environmental conditions:
- Chick embryo: chorioallantoic membrane (CAM)
- Fresh, fertile, White Leghor, eggs obtained from Avian Services in Frenchtown, New Jersey
- Acclimation: 7 d at 13°C before incubation
- Incubation: ca. 37°C with a relative humidity of 60 - 70% for 10 d (in a Kuhl incubator)
Vehicle:
water
Remarks:
10% test substance in water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
Test substance: 0.3 mL if liquid or 0.3 g if solid
Different concentrations: 1, 5 and 10% (Actual concentrations: 0.5, 2.5 and 5%)
Duration of treatment / exposure:
20 s
Duration of post- treatment incubation (in vitro):
Up to 5 minutes
Number of animals or in vitro replicates:
4
Details on study design:
After washing with physiological saline, membranes were observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects.
Irritation parameter:
other: mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis
Run / experiment:
Test substance - 1, 5 and 10% (Actual: 0.5, 2.5 and 5%)
Value:
>= 2.5 - <= 3.25
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: irritation potential: practically none
Other effects / acceptance of results:
CAM were exposed to the test substance at concentrations of 0, 1, 5 and 10% and the mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis were 1.75, 2.50, 3.25 and 3.00, respectively. The test substance was considered to have almost no irritation potential to CAM.

Results

 

Reference substance (%)

CAM

Scores

0.5 min

2 min

5 min

Total

Distilled water (100%)

1

0

0

0

0

2

0

0

1

1

3

0

3

0

3

4

0

3

0

3

Average

1.75

 

Test substance (%)

CAM

Scores

0.5 min

2 min

5 min

Total

Test substance (5% dd water) (Actual concentration: 0.5%)

1

0

3

0

3

2

0

3

0

3

3

0

3

0

3

4

0

0

1

1

Average

2.5

 

Test substance (%)

CAM

Scores

0.5 min

2 min

5 min

Total

Test substance (5% dd water) (Actual concentration: 2.5%)

1

0

0

1

1

2

0

3

3

6

3

0

3

0

3

4

0

3

0

3

Average

3.25

 

Test substance (%)

CAM

Scores

0.5 min

2 min

5 min

Total

Test substance (5% dd water) (Actual concentration: 5%)

1

0

3

0

3

2

0

3

0

3

3

0

3

0

3

4

0

3

0

3

Average

3

 

Discussion

Previous studies have shown that the CAM of the hen's egg is more sensitive to liquid irritants than is the rabbit eye. Therefore, the CAM results for the test substance at a specific concentration equate to Draize results for the test substance at two times that concentration. For example, Johnson's Baby Shampoo, when dosed at 50% in the CAM assay, elicits an average score of approximately 11. This result would correlate to the Draize score of approximately 10 to 15 that would be elicited by Johnson's Baby Shampoo, when dosed at 100%.

 

Conclusion

Under the conditions of this test, the results indicate that the test substance (5% dd water) at 1, 5 and 10% (actual concentrations of test substance 0.5, 2.5 and 5%) would have practically no irritation potential in vivo (The CAM results for the test substance at 0.5%, 2.5% and 5% are equivalent to Draize results for the test substance at 1%, 5% and 10 %).

Conclusions:
Under the study conditions, the test substance was considered to have almost no irritation potential to eye (HET-CAM).

Executive summary:

An study was conducted to determine the in vitro eye irritation potential of the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE' according to the HET-CAM test, in compliance with GLP. Chorioallantoic membrane (CAM) were exposed for 20 s to 0.3 mL or 0.3 g test substance at concentrations of 0 (vehicle only: distilled water), 1, 5 and 10% (Actual concentration 0.5, 2.5 and 5%). After exposure period, membranes were washed with physiological saline and observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects. Mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis were recorded and gave values of 1.75, 2.50, 3.25 and 3.00 for the concentrations 0, 1, 5 and 10%, respectively. Under the study conditions, the test substance was considered to have practically no irritation potential to eye at test concentrations up to 5% (CPT, 2003).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

Study 1 – Skin corrosion:

A study was conducted to determine the in vitro skin corrosion potential of the test substance, 'mono- and di- C16 -18 PSE and C16-18 AE20 PSE' using reconstructed human epidermis (RHE), according to a method similar to OECD Guideline 431, in compliance with GLP. Three replicates of 25 µg of the test substance (with 25 µL of distilled water), 50 µL of the negative control (water) and 8N positive control substance (8N Potassium hydroxide) were added to millicells MatTek Epiderm tissue samples for 3 minutes and 1 h time period. After exposure periods, the tissues were rinsed with phosphate buffered saline (PBS) to remove any residual material. After all tisses had been rinsed and dosed, they were placed for MTT assay. The MTT plates were then incubated for 3 h (at 37 ºC, 5% C02). After incubation period, the optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. The mean viability of cells exposed to the test substance were 101% and 99% of the negative control after 3 min and 1 h, respectively. The test substance did not reduce the viability to 50% or below and should be considered as non-corrosive to the skin. Under the study conditions, the test substance tested was determined to be non-corrosive to the skin (CPT, 2003).

Study 2 - Skin corrosion - read across study:

An in vitro study was conducted to determine the skin corrosion potential of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using Reconstructed Human Epidermis (RHE) cells, according to OECD 431 Guideline, 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 (25 mg) and reference substances (25 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 test substance and the reference substances were rinsed off from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT on Day 2. On Day 3, 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 substance were determined to be 103.7% and 93.8%, 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, 2017). Based on the results of the read across study, the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', is considered to be non-corrosive to the skin.

Study 3 - Skin irritation - read across study:

An in vitro study was conducted to determine the skin irritation potential of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE' using Reconstructed Human Epidermis (RHE) cells, according to OECD 439 Guideline, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, 95% RH. On Day 1, the tissues in triplicate were exposed to nominal 25 mg of test substance and 30 µL reference substances, applied topically for 60 ±1 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, 95% RH), followed by rinsing steps and a 42 ± 4 h post-dose incubation at 37°C, 5% CO2, 95%RH). On Day 2, the medium was changed and on Day 3, MTT viability test with readings at 570 nm without reference filter was performed. 30 µL of DPBS and 5% SDS were used as negative control and positive control, respectively. Viability of the tissues was assessed in MTT test and compared to the negative control. The percentage of viability obtained with the substance was 80.6%, which is well above the irritant limit of 50%. The study met all the validity criteria. Under the study conditions, the read across substance was determined be non-irritant to skin (XCellR8, 2017). Based on the results of the read across study, the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE' is considered to be non-irritating to the skin.

Further, as per a HERA 2009 review report, AEs with varying carbon chain lengths and ethoxylation degree were found to be slightly to severely irritating to skin in rabbits and rats. There was a trend observable that the degree of ethoxylation impacted the skin irritation potential of AE’s. AEs with lower ethoxylation degree (i.e., 1 -3 EO-units) appeared to be more irritating than AE’s with more than 4 ethoxy units (HERA, 2009).

Overall, based on the available weight of evidence, test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE', can be considered to be non-irritating to skin.

Eye: 

Study 1 – Eye corrosion:

An in vitro study was conducted to determine the eye irritation potential of the the test substance, ‘mono- and di- C16-18 PSE and C16-18 AE20 PSE’, using the Bovine Corneal Opacity and Permeability (BCOP) method, according to the OECD Guideline 437 and EU Method B.47, in compliance with GLP. The test substance was applied to test system at a concentration of 20% w/v in 0.9% w/v sodium chloride for 240 minutes followed by post exposure period at 32 ± 1ºC for 90 minutes. Negative and positive control substances were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The test substance IVIS was determined to be 89.1, which is well above the corrosive limit of 55. Therefore, the test substance was classified as Category 1 (irreversible effects on the eye) based on GHS/EU CLP criteria. The positive control IVIS was 127, which was outside the range of 65.1 to 123.3, however, as the score was only marginally exceeded, study author decided that this result was acceptable as the positive control group was still providing its intended function which is to show the sensitivity of the test system to a known ocular irritant. Therefore, this deviation was considered to have not affected the integrity or validity of the study. The negative control gave opacity of ≤1.3 and permeability ≤0.004, therefore the negative control acceptance criteria were satisfied. The test was considered to pass all the validity criterias. Under study conditions, the test substance was considered to be corrosive and classified as Eye Damage 1 (causes serious eye damage) based on EU CLP criteria (Envigo, 2017).

 

Study 2 – Eye irritation:

An study was conducted to determine the in vitro eye irritation potential of the test substance, 'mono- and di- C16-18 PSE and C16-18 AE20 PSE' according to the HET-CAM test, in compliance with GLP. Chorioallantoic membrane (CAM) were exposed for 20 s to 0.3 mL or 0.3 g test substance at concentrations of 0 (vehicle only: distilled water), 1, 5 and 10% (Actual concentration 0.5, 2.5 and 5%). After exposure period, membranes were washed with physiological saline and observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects. Mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis were recorded and gave values of 1.75, 2.50, 3.25 and 3.00 for the concentrations 0, 1, 5 and 10%, respectively. Under the study conditions, the test substance was considered to have practically no irritation potential to eye at test concentrations up to 5% (CPT, 2003).

Based on the results of the BCOP assay, the test substance, ‘mono- and di- C16-18 PSE and C16-18 AE20 PSE' is considered to be corrosive to eyes.

Justification for classification or non-classification

Skin irritation:

Based on the available weight of evidence from in vitro studies with the test and read across substances, the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE' warrants a 'Skin Irrit. 2; H315 - Causes skin irritation' classification according to EU CLP criteria (Regulation 1272/2008/EC).

Eye irritation:

Based on the available weight of evidence from in vitro studies with the test and read across substances, the test substance, 'mono- and di- C18 -unsatd. PSE and C18 -unsatd. AE5 PSE', warrants a 'Eye damage 1; H318- Causes serious eye damage’ classification according to EU CLP criteria (Regulation 1272/2008/EC). Labelling for this endpoint should include “Danger” as signal word.