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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- C16-18 PSE and C16-18 AE10 PSE', is considered to be non-irritating to the 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 corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 16, 2017 to June 15, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (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# 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: CLP criteria not met
Remarks:
(not corrosive)
Conclusions:
Under the study conditions, the test substance was determined to be non-corrosive to the skin.
Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the test 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 test 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 test substance was determined to be non-corrosive to the skin (XCellR8, 2017).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 18, 2017 to June 23, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
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. QA statement)
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:
Under the study conditions, the test substance was determined to be non-irritating to the skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test 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 test 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 test substance was determined be non-irritating to the skin (XCellR8, 2017).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
weight of evidence
Study period:
July 4, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
BCOP test was performed to identify test substance that can induce serious eye damage and to identify test substance not requiring classification for eye irritation or serious eye damage.
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 prepared immediately on arrival.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
20% w/v test substance in sodium chloride 0.9% w/v
Duration of treatment / exposure:
240 minutes
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 preparation 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 ± 1ºC for 240 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 negative and positive control data was shared with Envigo - Shardlow study number NX25RF and XL29CC.

Application of sodium fluorescein
Following the 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 (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC 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. 51.1
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 measurements

Treatment

Cornea Number

Opacity

Permeability (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

13

3

74

71

69.7

0.008

0.004

 

14

2

38

36

34.7

0.011

0.007

 

15

3

53

50

48.7

0.000

0.000

 

 Mean

 

 

 

51.0

 

0.004

51.1

OD= Optical density            

* = Control data shared with Envigo - Shardlow study number NX25RF and XL29CC

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

13

Cloudy

14

Cloudy

15

Cloudy

*= Control data shared with Envigo - Shardlow study number NX25RF and XL29CC

 

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

 

Treatment

In Vitro Irritancy Score

Test substance

51.1

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 was reported as a deviation. The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

Deviation

The positive control group had an overall IVIS of 127.0. This 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.

Interpretation of results:
other: CLP - inconclusive results
Conclusions:
Under the study conditions, no prediction of eye irritation for the test substance could be made.
Executive summary:

A study was performed to determine the eye irritation potential of test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using BCOP test, according to EU Method B.47 and OECD Guideline 437, in compliance with GLP. Appropriately prepared bovine cornea, in triplicates were expsured to 0.75 mL of the test substancs (20% w/v in sodium chloride 0.9% w/v ) and reference substances (Negative control: sodium chloride 0.9% w/v, Positive control: Imidazole 20% w/v solution in sodium chloride 0.9% w/v). 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 240 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 Eagle’s Minimum Essential Medium (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 negative and positive control data was shared with Envigo - Shardlow study number NX25RF and XL29CC. 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 test substance, negative control and positive control were reported to be 51.1, 1.4 and 127 respectively. The study has met all validity criteria, except for positive 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 test substance, which is >3 and <55%, no prediction of eye irritation potential could be made (Envigo, 2017).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From August 29, 2017 to August 31, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This in vitro risk assessment assay predicts the acute eye irritation potential of a chemical by measurement of its cytotoxic effect on the EpiOcular™ corneal epithelial model.
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: EpiOcularTM tissue model (OCL-200-MatTek Corporation)
Strain:
other: Keratinocyte 4F1188
Details on test animals or tissues and environmental conditions:
Test system
The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo. MatTek’s EpiOcularTM system consists of normal, human-derived keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure with progressively stratified, but not cornified cells which closely parallel the corneal epithelium (for information see www.mattek.com).

QC results for the specific lot of models received (Lot# 27002) were checked in-house for MatTek acceptance ranges with the following outcome:
- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 0.3 % Triton X-100) where ET50 is the time taken for 0.3 % 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



Vehicle:
other: PBS (Sterile Dulbecco’s Phosphate Buffered Saline)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
20 µL of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) plus 50 mg of test substance.
Duration of treatment / exposure:
6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion
Duration of post- treatment incubation (in vitro):
18 h ± 15 minutes post-treatment incubation
Number of animals or in vitro replicates:
Three tissues per condition (n=3)


Irritation parameter:
other: Percentage of viability (relative to negative control)
Value:
12.922
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Prior to the study, the required compatibility checks (as per SOP L0069) confirmed that the test substance did not interfere with MTT or solvent.

Results

Table 1: Viability measurements after 6 h (± 15 min) of application and 18 h (± 15 min) post-incubation of test and reference substances.

Condition

Tissue #

Raw data

Blank corrected data

Mean OD

% of viability

Aliquot 1

Aliquot 2

Aliquot 1

Aliquot 2

NC

Tissue 1

2.049

2.094

1.869

1.914

1.891

106.635

Tissue 2

1.83

1.801

1.650

1.621

1.635

92.200

Tissue 3

1.956

1.993

1.776

1.813

1.794

101.165

PC

Tissue 1

0.839

0.847

0.659

0.667

0.663

37.365

Tissue 2

0.782

0.781

0.602

0.601

0.601

33.897

Tissue 3

0.696

0.716

0.516

0.536

0.526

29.640

TA3

Tissue 1

0.269

0.261

0.089

0.081

0.085

4.774

Tissue 2

0.417

0.449

0.237

0.269

0.253

14.247

Tissue 3

0.541

0.52

0.361

0.340

0.350

19.744

NC: negative control (sterile H2O), PC: Positive control (neat Methyl Acetate), TA3: Test substance

Table 3: Mean and SD of viability measurements and of viability percentages after 6 h (± 15 min) of application and 18 h (± 15 min) post-incubation.

Name

Code

Mean of OD

SD of OD

Mean of viability (%)

SD of viability (%)

CV %

Classification

Sterile water

NC

1.774

0.129

100.000

7.288

7.288

Non-Irritant

Methyl Acetate

PC

0.597

0.069

33.634

3.869

11.504

Irritant

Test substance

TA3

0.229

0.134

12.922

7.573

58.604

Irritant

 

Table 3: Results Summary

Test Substance

Percentage of viability
(relative to negative control)

Classification
(Irritant (I)/Non-Irritant (NI)

Test substance

12.922%

Irritant (I)

 

The test substance reduced the viability to below 60% after 6 h ± 15 minutes of application and should be considered as Irritant to the eye.

Acceptance criteria

Criteria

Description

Actual values

PASS/FAIL

Acceptance criterion 1

The mean OD570 of the negative control (treated with sterile water) tissues is > 0.8 and < 2.5.

1.774

PASS

Acceptance criterion 2

The mean of the positive control relative percentage viability is below 50% of negative control viability after 6 h ± 15mins exposure.

33.634

PASS

Acceptance criterion 3

The SD between three tissues replicates should not exceed 18% in the same run (for negative and positive control tissues and tissues of test substances).

NC: 7.288

PC: 3.869

Test Substance: 7.573

 

PASS

Interpretation of results:
other: Category 1 or Category 2 (Irritant to the eye) based on EU CLP criteria
Remarks:
Further categorization needed
Conclusions:
Under the study conditions, the test substance was determined to be irritant to the eye.
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C16 -18 PSE and C16 -18 AE10 PSE', using Reconstructed human cornea-like Epithelium (RhCE), according to OECD Guideline 492, in compliance with GLP. EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH. On Day 1, after pre-wetting tissues with 20 µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, the tissues in triplicate were exposed to single topical application of 50 mg neat test substance or 50 µL of reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation, prior to the MTT endpoint. On Day 2, MTT tests and measurement were performed 570 nm without reference filter. The viability of the tissues were assessed and compared to a negative control. The percentage viability obtained with the test substance was determined to be 12.922%, which is well below the threshold (i.e., >60%) indicating no irritation potential. Therefore, the study authors concluded the test substance as irritant to the human eye under the study conditions (XCELLR8, 2017). However, based on the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage), hence, the classification for the test substance is considered to be inconclusive.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
July 04, 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 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:
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:
Based on results of the read across study, the test substance, mono- and di- C16-18 PSE + mono- and di- C16-18 PSE, EO-10 is considered as inducing serious eye damage and classified as Category 1 (irreversible effects on the eye) based on EU CLP criteria.
Executive summary:

A study was conducted to determine the eye irritation potential of the the read across 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 read across 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 read across substance IVIS was determined to be 89.1, which is well above the corrosive limit of 55. Therefore, the read across 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 read across substance was considered to be corrosive and classified as Eye Damage 1 (causes serious eye damage) based on EU CLP criteria (Envigo, 2017). Based on the results of the read across study, similar corrosive conclusion can be considered for the test substance, 'mono- and di- C16 -18 PSE and C16 -18 AE10 PSE'.

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

Additional information

Skin irritation:

Study 1: An in vitro study was conducted to determine the skin corrosion potential of the test 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 test 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 test substance was determined to be non-corrosive to the skin (XCellR8, 2017).

Study 2: An in vitro study was conducted to determine the skin irritation potential of the test 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 test 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 test substance was determined be non-irritating to the skin (XCellR8, 2017).

Eye irritation:

Study 1: A study was performed to determine the eye irritation potential of test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using BCOP test, according to EU Method B.47 and OECD Guideline 437, in compliance with GLP. Appropriately prepared bovine cornea, in triplicates were expsosed to 0.75 mL of the test substances (20% w/v in sodium chloride 0.9% w/v ) and reference substances (Negative control: sodium chloride 0.9% w/v, Positive control: Imidazole 20% w/v solution in sodium chloride 0.9% w/v). 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 240 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 Eagle’s Minimum Essential Medium (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 negative and positive control data was shared with Envigo - Shardlow study number NX25RF and XL29CC. 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 test substance, negative control and positive control were reported to be 51.1, 1.4 and 127 respectively. The study has met all validity criteria, except for positive 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 test substance, which is >3 and <55%, no prediction of eye irritation potential could be made (Envigo, 2017).

Study 2: An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C16 -18 PSE and C16 -18 AE10 PSE', using Reconstructed human cornea-like Epithelium (RhCE), according to OECD Guideline 492, in compliance with GLP. EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH. On Day 1, after pre-wetting tissues with 20 µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, the tissues in triplicate were exposed to single topical application of 50 mg neat test substance or 50 µL of reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation, prior to the MTT endpoint. On Day 2, MTT tests and measurement were performed 570 nm without reference filter. The viability of the tissues were assessed and compared to a negative control. The percentage viability obtained with the test substance was determined to be 12.922%, which is well below the threshold (i.e., >60%) indicating no irritation potential. Therefore, the study authors concluded the test substance as irritant to the human eye under the study conditions (XCELLR8, 2017). However, based on the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage), hence, the classification for the test substance is considered to be inconclusive.

Study 3: A study was conducted to determine the eye irritation potential of the read across 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 read across 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 read across substance IVIS was determined to be 89.1, which is well above the corrosive limit of 55. Therefore, the read across 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 read across substance was considered to be corrosive and classified as Eye Damage 1 (causes serious eye damage) based on EU CLP criteria (Envigo, 2017).

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. No relationship could be established between the chemical structures of the tested AEs and their eye irritation responses (HERA, 2009).

In absence of clear conclusions in the in vitro tests with the test substance and based on the results of the in vitro read across study, which differs only in the number of ethoxylation (i.e., 20EO vs 10EO), the test substance, 'mono- and di- C16 -18 PSE and C16 -18 AE10 PSE' can be considered to be corrosive to eyes (in the worst case).

Justification for classification or non-classification

Skin irritation:

Based on the results of in vitro skin irritation/corrosion studies, the test substance, ‘mono- and di- C16-18 PSE and C16-18 AE10 PSE’, is concluded not to warrant skin irritation classification according to the EU CLP criteria (Regulation 1272/2008/EC).

 

Eye irritation:

Based on the results of read across in vitro eye irritation study (BCOP), the ‘mono- and di- C16-18 PSE and C16-18 AE10 PSE’ is concluded to warrant ‘Eye damage 1: H318- causes serious eye damage’ classification according to the EU CLP criteria (Regulation 1272/2008/EC). Labelling for this endpoint should include “Danger” as signal word.