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Diss Factsheets

Administrative data

Description of key information

Based on the results of the studies with the test substance and/or the two main constituents, the test substance, 'mono- and di- C16 PSE, K+ and H3PO4', is considered to be non-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:
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:
Deviation was not considered to have affected the integrity or interpretation of the results
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
MatTek EpiDermTM tissue model EPI-200
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:
25 mg after pre-wetting with 25 uL of DPBS
Duration of treatment / exposure:
60 ± 1 minutes of treatment
Duration of post-treatment incubation (if applicable):
42 ± 4 h
Number of replicates:
3 replicates for the test substance, positive and negative control
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test substance
Value:
ca. 93.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control
Value:
ca. 3.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control
Value:
ca. 100
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.

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

TA2

Tissue 1

2.005

2.027

1.838

1.860

1.849

100.1

Tissue 2

2.008

1.821

1.841

1.654

1.747

94.6

Tissue 3

1.698

1.799

1.531

1.632

1.581

85.6

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

Note: Rounded figures used.

Table 2: Mean and SD of cell viability measurements and of viability percentages after a 60 ±1 min 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

TA2

1.726

0.135

93.5

7.31

7.83

Non-Irritant

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

Note: Rounded figures used.

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%

TA2: 7.31%

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

- 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%.

- 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 93.5%, therefore it is considered as non-Irritant to the skin.

Interpretation of results:
other: CLP
Remarks:
non-irritant based on CLP criteria
Conclusions:
Under the study conditions, the test substance was determined to be non-irritant to skin.
Executive summary:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100%), using Reconstructed Human Epidermis (RHE) method, according to OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative controls for 60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5 % CO2, 95 % RH) and 42 h post incubation period. Test was performed with 3 replicates for each type of treatment. Tissues were first pre-wetted with 25 μL DPBS (Sterile Dulbecco’s Phosphate Buffered Saline), subsequently 25 mg (nominal) of the neat test substance was applied. 30 μL of DPBS was used as negative control and 5% of sodium dodecyl sulphate (SDS) as positive control. 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 89.1%, 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 to be non-irritating to skin (XCellR8, 2017).

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)
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 for test substance, negative and positive controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
100.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
103
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

 

 

 

TA2

1

1.635

1.656

1.645

1.754

94.1

100.3

5.8

5.8

2

1.741

1.801

1.771

 

101.3

 

 

 

3

1.811

1.883

1.847

 

105.6

 

 

 

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), TA2: Test substance

Table 2: Cell viability measurements after 1 h of application

Name

Tissue n°

1h endpoint

 

Aliq. 1

Aliq. 2

mean

OD Mean

viability

Mean

SD

CV

[%]

[%]

[%]

[%]

NC

1

1.842

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

 

 

 

TA2

1

1.860

1.885

1.872

1.831

105.3

103.0

2.0

1.9

2

1.816

1.817

1.816

 

102.2

 

 

 

3

1.802

1.811

1.806

 

101.6

 

 

 

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), TA2: Test substance.

 

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

 

3min

1hour

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

TA2

100.3

5.8

5.8

103.0

2.0

1.9

PC

18.1

4.1

22.7

14.0

3.3

23.3

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

 

Table 4: Results Summary

Test substance

Test Substance ID

Viability after 3 minutes application

(% to negative control)

Viability ≥ 50% after 3 min (Yes/No)

Viability after 1h application

(% to negative control)

Viability ≥ 15% after 1h (Yes/No)

Corrosive (C)/Non corrosive(NC)

Test substance

TA2

100.3%

Yes

103.0%

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

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 hour 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

TA2: 5.8% after 3 min, 1.9% 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: Not classified based on EU CLP criteria
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 PSE, K+ and H3PO4' (purity: 100%), using Reconstructed Human Epidermis (RHE) Method, according to OECD Guideline 431, in compliance with GLP. EpiDermTM tissues were kept overnight at 4°C. On Day 1, the tissues were pre-incubated for 1 h at 37°C, 5% CO2, 95% RH. After incubation, tissues were exposed 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 100.3% and 103%, 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 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:
key study
Study period:
From June 29, 2017 to August 10, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
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 positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL test substance or reference substances (Positive control: 20% w/v imidazole solution in sodium chloride 0.9% w/v, Negative control: Sodium chloride 0.9% w/v)
Duration of treatment / exposure:
At 32 ± 1 ºC for 240 minutes
Duration of post- treatment incubation (in vitro):
At 32 ± 1 ºC for 90 minutes for permeablity assessment
Number of animals or in vitro replicates:
Three corneas to each test substance and reference substances
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 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
 
Selection of corneas and opacity reading
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. 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 ± 1 ºC 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.

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 ± 1 ºC for 90 minutes.
 
Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
 
Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin. In this study histopathology was not required.
 
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.
Irritation parameter:
in vitro irritation score
Run / experiment:
Test substance
Value:
ca. 5.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Irritation parameter:
in vitro irritation score
Run / experiment:
Positive control
Value:
ca. 99.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative control
Value:
ca. 1.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied. 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 following table 1:

Treatment

Cornea Number

Opacity

Permeability (Optical density)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative Control

1

3

3

0

 

0.004

 

 

2

3

7

4

 

0.003

 

 

3

4

4

0

 

0.002

 

 

Mean

 

 

1.3

 

0.003

 

1.4

Positive
Control

4

4

77

73

71.7

1.355

1.352

 

5

5

89

84

82.7

1.072

1.069

 

6

5

82

77

75.7

2.125

2.122

 

Mean

 

 

 

76.7

 

1.514

99.4

Test Substance

7

2

10

8

6.7

0.077

0.074

 

8

2

11

9

7.7

0.014

0.011

 

9

4

6

2

0.7

0.008

0.005

 

Mean

 

 

 

5.0

 

0.030

5.5

 

Corneal Epithelium Condition

The condition of each cornea is given in below table 2:

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

Partly Cloudy

8

Partly Cloudy

9

Partly Cloudy

The corneas treated with the test substance were partly 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

5.5

Negative Control

1.4

Positive Control

99.4

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

Conclusion

No prediction of eye irritation can be made

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

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100 %), using Bovine Corneal Opacity Test (BCOP), according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (20% w/v in sodium chloride 0.9% w/v), negative control (Sodium chloride 0.9% w/v) and positive control (20% w/v Imidazole solution in sodium chloride 0.9% w/v) substances 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. A post treatment opacity reading was taken and each cornea was visually observed. 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. 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 positive control IVIS was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied. The negative control resulted in opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 5.5, therefore, no prediction of eye irritation could be made for the test substance. Under the study conditions, no prediction of eye irritation could be made for the test substance (Envigo, 2018).

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 with acceptable restrictions
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:
Deviations were 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:
Deviations were 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 positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL test substance or reference substances (Positive control: 20% w/v imidazole solution in sodium chloride 0.9% w/v, Negative control: Sodium chloride 0.9% w/v)
Duration of treatment / exposure:
At 32 ± 1 ºC for 240 minutes
Duration of post- treatment incubation (in vitro):
At 32 ± 1 ºC for 90 minutes for permeablity assessment
Number of animals or in vitro replicates:
Three corneas to each test substance and reference substances
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 ± 1 ºC 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 ± 1 ºC 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 study number NX25RF and LM55TK.
 
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 ± 1 ºC for 90 minutes.
 
Permeability determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.360 µL of media representing each cornea was dispensed into the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
 
Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre‑labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin. In this study histopathology was not required.
 
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.
Irritation parameter:
in vitro irritation score
Run / experiment:
Test substance
Value:
ca. 77
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Positive control
Value:
ca. 127
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative control
Value:
ca. 1.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
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.

Results

Corneal Opacity and Permeability measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in following table 1:

Treatment

Cornea number

Opacity

Permeability (OD492)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Treatment-Pre‑Treatment

Corrected Value

 

Corrected Value

Negative ControlÅ

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
Å

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

10

2

74

72

70.7

0.007

0.003

 

11

2

82

80

78.7

0.000

0.000

 

12

3

86

83

81.7

0.000

0.000

 

Mean

 

 

 

77.0

 

0.001

77.0

 

Corneal Epithelium Condition

The condition of each cornea is given in below table 2:

Treatment

Cornea number

observation
post treatment

Negative Control*

1

Clear

2

Clear

3

Clear

Positive Control*

4

Cloudy

5

Cloudy

6

Cloudy

Test Substance

10

Cloudy

11

Cloudy

12

Cloudy

* = Control data shared with Envigo study number NX25RF and LM55TK

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

77.0

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

Category 1. UN GHS or EU CLP Causes serious eye damage

Interpretation of results:
other: Category 1 (irreversible effects on the eye) based on EU CLP criteria
Conclusions:
Based on the results of the read across study, the test substance, mono- and di- C16 PSE, K+ and H3PO4 is considered to be EU CLP category 1 eye corrosive (H318: Causes serious eye damage).
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the read across substance, 'mono- C16 PSE and C16-OH' (purity: 100%), using Bovine corneal Opacity Test (BCOP), according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (20% w/v in sodium chloride 0.9% w/v), negative control (Sodium chloride 0.9% w/v) and positive control (20% w/v Imidazole solution in sodium chloride 0.9% w/v) substances 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 completeEagle’sMinimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The negative and positive control data was shared with Envigo study number LM55TK and NX25RF. 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. 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 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, the 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. The negative control gave opacity of ≤2.4 and permeability ≤0.072, the negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 77, which is well above the threshold for corrosive classification. Under the study conditions, the read across substance was considered to be corrosive and classified as Category 1 or Eye Damahge 1-H318: causes serious eye damage, according to UN GHS or EU CLP (Envigo, 2018). Based on the results of the read across study, a similar conclusion can be considered for the test substance, 'mono- and di- C16 PSE, K+ and H3PO4'.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1975 to 1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: Federal Hazardous Substances Act, 21 CFR, 191.12 (1964)
Deviations:
not specified
GLP compliance:
not specified
Remarks:
The actual study conducted was conducted in 1975
Species:
rabbit
Strain:
New Zealand White
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
0.1 mL of commercial preparations (undiluted) of phosphoric acid of varying concentration were used (75, 80 and 85% aqueous solutions)
Duration of treatment / exposure:
24 h and 1 minute
Observation period (in vivo):
14 d
Number of animals or in vitro replicates:
3
Details on study design:
0.1 mL of commercial preparations of phosphoric acid of varying concentration (75, 80 and 85% aqueous solutions) were placed into the conjunctival sac of New Zealand albino rabbits. Eye responses were scored in accordance with the Federal Hazardous Substances Act, 21 CFR, 191.12 (1964).
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 40.1
Max. score:
110
Reversibility:
fully reversible within: 14 d: All scored zero
Remarks on result:
positive indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 4.44
Max. score:
10
Reversibility:
fully reversible within: 14 d
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 16.11
Max. score:
20
Reversibility:
fully reversible within: 14 d
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 16.11
Max. score:
20
Reversibility:
fully reversible within: 14 d
Remarks on result:
other: Score presented as total conjuctivae grading
Remarks:
Covered in conjuctivae grading
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
ca. 21.66
Max. score:
80
Reversibility:
fully reversible within: 14 d
Remarks on result:
other: Score presented as total cornea score grading
Remarks:
Covered in cornea score grading
Irritant / corrosive response data:
Irritant/corrosive response data
Immediate: Discomfort was severe with thrashing about the stocks and eyes tightly closed.
10 minutes: Areas of barely perceptible to slight corneal cloudiness, iris reaction to light was sluggish, severe erythema, very slight to slight edema, and copious discharge.
1 h: Areas of slight corneal cloudiness, iris showed sluggish reaction to light, severe erythema (necrosis), slight edema, and copious discharge.
24-168 h: Gradual improvement.
14 d: all scored zero.

Results

Eye irritation response scoring

Hours

Structure

 

Animals number

 

Mean score (X/110)

1

2

3

1

Cornea

45

30

30

56

Iris

5

5

5

Conjuctivae

16

16

16

24

Cornea

30

30

30

52.3

Iris

5

5

5

Conjuctivae

18

18

16

48

Cornea

30

20

15

42.6

Iris

5

5

5

Conjuctivae

16

16

16

72

Cornea

20

10

10

25.6

Iris

5

0

0

Conjuctivae

12

12

8

120

Cornea

10

10

10

20

Iris

0

0

0

Conjuctivae

12

10

8

168

Cornea

10

0

5

13.6

Iris

0

0

0

Conjuctivae

10

8

8

Interpretation of results:
other: Corrosive to the eye based on Federal Hazardous Substances Act, 21 CFR, 191.12 (1964) criteria
Conclusions:
Under the study conditions, the test substance was considered to be corrosive to rabbit eyes.
Executive summary:

An in vivo study was conducted to determine the eye irritation potential of the test substance, phosphoric acid (75 -85% aqueous solution) in rabbits, according to Federal Hazardous Substances Act, 21 CFR, 191.12 (1964). In the study, 0.1 mL of neat commercial preparations of phosphoric acid were placed into the conjunctival sac of three New Zealand albino rabbits for up to 24 h. Eye responses were scored 1, 24, 48, 72, 120 and 168 h after exposure in accordance with the Federal Hazardous Substances Act, 21 CFR, 191.12 criteria. At 1 h, areas of slight corneal cloudiness, sluggish reaction of iris to light, severe erythema (with necrosis), slight edema, and copious discharge were observed. However, during 24 to 168 h observation period, gradual improvement were noted in all animals and by 14 d all effects were seen to be reversed (all scores were zero). The overall mean irritation score was calculated to be 40.1 for 24/48/72 h observations. Under the study conditions, the test substance was considered to be corrosive to rabbit eyes (OECD SIDS, 2009).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

Skin corrosion (RHE) study: An in vitro study was conducted to determine the skin corrosion potential of the test substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100%), using Reconstructed Human Epidermis (RHE) Method, according to OECD Guideline 431, in compliance with GLP.EpiDermTM tissues were kept overnight at 4°C. On Day 1, the tissues were pre-incubated for 1 h at 37°C, 5% CO2, 95% RH. After incubation, tissues were exposed 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 100.3% and 103%, 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 skin (XCellR8, 2017).

Skin irritation (RHE) study:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100%), using Reconstructed Human Epidermis (RHE) method, according to OECD Guideline 439, in compliance with GLP.Three tissues of the human skin model EpiDermTM were treated with the test substance, positive or negative controlsfor 60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5 % CO2, 95 % RH) and 42 h post incubation period.Test was performed with 3 replicates for each type of treatment.Tissues were first pre-wetted with 25 μL DPBS (Sterile Dulbecco’s Phosphate Buffered Saline), subsequently 25 mg (nominal) of the neat test substance was applied. 30 μL of DPBS was used as negative control and 5% of sodium dodecyl sulphate (SDS) as positive control. 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 89.1%, 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 to be non-irritating to skin (XCellR8, 2017).

Based on the available in vitro study results, the test substance is considered to be non-irritating to skin.

Eye:

In absence of clear conclusion from available BCOP study with the test substance, the endpoint has been assessed based on in vitro/in vivo studies available on substances representative of the main constituents, which can be categorised as phosphate esters (PSE) and free phosphoric acid (H3PO4). The results are presented below:

Eye corrosion (BCOP) study:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100 %), using Bovine Corneal Opacity Test (BCOP), according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (20% w/v in sodium chloride 0.9% w/v), negative control (Sodium chloride 0.9% w/v) and positive control (20% w/v Imidazole solution in sodium chloride 0.9% w/v) substances 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. A post treatment opacity reading was taken and each cornea was visually observed. 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. 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 positive control IVIS was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied. The negative control resulted in opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 5.5, therefore, no prediction of eye irritation could be made for the test substance. Under the study conditions, no prediction of eye irritation could be made for the test substance (Envigo, 2018).

Constituent 1: PSE (read across study):

An in vitro study was conducted to determine the eye irritation potential of the read across substance, 'mono- C16 PSE and C16-OH' (purity: 100%), using Bovine corneal Opacity Test (BCOP), according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (20% w/v in sodium chloride 0.9% w/v), negative control (Sodium chloride 0.9% w/v) and positive control (20% w/v Imidazole solution in sodium chloride 0.9% w/v) substances 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 completeEagle’sMinimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. The negative and positive control data was shared with Envigo study number LM55TK and NX25RF. 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. 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 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, the 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. The negative control gave opacity of ≤2.4 and permeability ≤0.072, the negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 77, which is well above the threshold for corrosive classification. Under the study conditions, the read across substance was considered to be corrosive and classified as Category 1 or Eye Damahge 1-H318: causes serious eye damage, according to UN GHS or EU CLP (Envigo, 2018).

Constituent 2: phosphoric acid:

An in vivo study was conducted to determine the eye irritation potential of the test substance, phosphoric acid (75 -85% aqueous solution) in rabbits, according to Federal Hazardous Substances Act, 21 CFR, 191.12 (1964). In the study, 0.1 mL of neat commercial preparations of phosphoric acid were placed into the conjunctival sac of three New Zealand albino rabbits for up to 24 h. Eye responses were scored 1, 24, 48, 72, 120 and 168 h after exposure in accordance with the Federal Hazardous Substances Act, 21 CFR, 191.12 criteria. At 1 h, areas of slight corneal cloudiness, sluggish reaction of iris to light, severe erythema (with necrosis), slight edema, and copious discharge were observed. However, during 24 to 168 h observation period, gradual improvement were noted in all animals and by 14 d all effects were seen to be reversed (all scores were zero). The overall mean irritation score was calculated to be 40.1 for 24/48/72 h observations. Under the study conditions, the test substance was considered to be corrosive to rabbit eyes (OECD SIDS, 2009).

Overall, based on the available weight of evidence information, the test substance, 'mono- and di- C16 PSE, K+ and H3PO4', is considered to be corrosive to the eyes.

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 PSE, K+ and H3PO4' does not warrant classification for skin irritation according to the EU CLP criteria (Regulation 1272/2008/EC).

 

Eye irritation:

Based on the results of ex vivo/in vivo eye irritation studies on substances representative of the main constituents, the test substance, 'mono- and di- C16 PSE, K+ and H3PO4' warrants an ‘Eye damage 1: H318- causes serious eye damage’ classification according to the EU CLP criteria (Regulation EC 1272/2008). Labelling for this endpoint should include “Danger” as signal word.