Alcohols, C18-unsatd and ethoxylated C18-unsatd., phosphates (5 moles ethoxylation)

Endpoint summary

Description of key information

Based on the available weight of evidence, the test substance, ‘mono- and di- C18-unsatd PSE and C18-unsatd AE5 PSE’, is considered to be irritating to skin and corrosive to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation EpiDermTM skin model in vitro toxicity testing system

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Lot no.: 146-100

Test system:

human skin model

Remarks:

artificial three-dimensional model of human skin

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Validated, accurate and reliable method for the prediction skin irritationg and no-label (no-skin irritating) test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL test substance, negative or positive control

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

cells after 3 min

Run / experiment:

MTT test (mean viability compared to the negative control - %)

Value:

78

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

cells after 1 h

Run / experiment:

MTT test (mean viability compared to the negative control - %)

Value:

91

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 test substance did not induce a significant decrease of tissue viability with values of 78 and 91% after 3 min and 1 h, respectively.
The positive control induced a significant decrease of tissue viability with values of 29 and 17% after 3 min and 1 h, respectively.

Interpretation of results:

other: CLP criteria for corrosion not met

Remarks:

non-corrosive

Conclusions:

Under the study conditions, the test substance (pH 6.20) did not induce a significant tissue mortality and was therefore predicted to be non-corrosive to skin.

Executive summary:

A study was conducted to determine the in vitro skin irritation potential of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd. AE5 PSE', using Reconstructed Human Epidermis (RHE) cells, according to a method similar to OECD Guideline 431, in compliance with GLP. Three replicates of 50 µL test substance (undiluted) were applied topically for 3 minutes or 1 h to the model skin surface. Distilled water and potassium hydroxide were used as negative and positive controls respectively. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. The time at which 50% percent viability was acheived (ET-50) was estimated for each tissue. The test substance did not induce a significant decrease of tissue viability with values of 78 and 91% after 3 min and 1 h, respectively. The positive control induced a significant decrease of tissue viability with values of 29 and 17% after 3 min and 1 h, respectively. Therefore, the study met the validity criteria. Under the study conditions, the test substance (at pH 6.20) was predicted to be non-corrosive to skin (CPT, 2004).

Reference 2

Endpoint:

skin irritation / corrosion, other

Remarks:

Corrositex test method

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2005

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: Corrositex test method

Deviations:

no

Principles of method if other than guideline:

Corrositex® test is based upon a biomembrane and chemical detection system, which becomes colored when exposed to potentially corrosive substances. The Corrositex® test is performed in three steps. First, a qualification test is done to insure that the test sample and the CDS reagent are compatible. If a physical change or color change is observed, the sample is judged to be compatible with the detection solution and the remainder of the test is performed. The second step of the Corrositex® test utilizes appropriate indicator solutions to permit categorization of the test sample as either a Corrositex® Category 1 or Corrositex® Category 2 material. Corrositex® Category 1 materials are typically strong acids/bases, while Corrositex® Category 2 materials are typically weak acids/bases. The third step in the test is performed by applying the test sample to the biobarrier. When the chemical permeates through or destroys the full thickness of this biobarrier, it comes into contact with the CDS which then undergoes a simple color change. This color change is visually observed and the time required for the color change to occur is recorded. Users simply record the time it takes for the sample to break through the membrane. Then, depending on their needs, they can assign the proper GHS Category and/or U.N. Packing Group classification for U.S. DOT or EPA compliance, or use the data as a ranking tool or to substantiate marketing claims.

GLP compliance:

not specified

Test system:

other: Corrositex test method

Vehicle:

unchanged (no vehicle)

Amount/concentration applied:

150 µL

Number of replicates:

4

Irritation / corrosion parameter:

other: Corrositex time (minutes)

Run / experiment:

Mean

Value:

> 60

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Non-corrosive

The test substance sample was compatible with the Corrositex system and was classified as a Category 2 material. The results for the 4 replicates were highly reproducible and a mean time of > 60 minutes was required to destroy the synthetic barrier.

Interpretation of results:

other: Non-corrosive; CLP criteria for corrosion not met

Conclusions:

under the study conditions, the test substance was considered non-corrosive (Corrositex test method).

Executive summary:

A study was conducted to determine the corrosive potential and to designate the Packing Group classification of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd. AE5 PSE' using the Corrositex test method. This test predicts the in vivo corrosive potential of a chemical compound or mixture by using an endpoint, i.e. the time it takes for the chemical to permeate through or destroy a synthetic biobarrier. When a chemical has passed through this biobarrier, a visual change is produced in a proprietary chemical detection system (CDS). The test is performed in 3 steps. A qualification test is done to insure that the test substance sample and the CDS reagent are compatible (observation of any changes). The appropriate indicator solutions are then selected in order to permit categorization of the sample as either a Category 1 or 2 material. Finally, the test substance is applied on the biobarrier. The changes and the time at which they appear, are recorded following visual observation. The test substance is then classified according to the occurence of the changes (Packing I (0 to 3 min), II (3 to 60 min), III (30 to 240 min) or non-corrosive (> 60 min), depending on the category). Four replicates were analysed in this experiment. The test substance sample was compatible with the Corrositex system and was classified as a Category 2 material. The results for the 4 replicates were highly reproducible and a mean time of > 60 minutes was required to destroy the synthetic barrier. Under the study conditions, the test substance was considered non-corrosive (IVI, 2005).

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

2003

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation EpiDermTM skin model in vitro toxicity testing system

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

not applicable

GLP compliance:

yes

Specific details on test material used for the study:

Lot no.: 117-860

Test system:

human skin model

Remarks:

artificial three-dimensional model of human skin

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Validated, accurate and reliable method for the prediction skin irritationg and no-label (no-skin irritating) test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

100 µL test substance, negative or positive control

Duration of treatment / exposure:

1, 4.5 and 20 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

cells after 1h

Run / experiment:

MTT test (mean viability compared to the negative control - %)

Value:

13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

cells after 4.5 and 20 h

Run / experiment:

MTT test (mean viability compared to the negative control - %)

Value:

3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The test substance induced a significant decrease of tissue viability with values of 13, 3 and 3% after 1, 4.5 and 20 h, respectively. The ET-50 value was determined to be less than 0.5 h.
The positive control also induced a significant decrease of tissue viability with values of 78, 37 and 5% after 1, 4.5 and 20 h, respectively. The ET-50 value was determined to be ca. 2.9 h, which was out of domain for QC acceptance criteria (4 to 8.7 h). Therefore, the study did not fulfill validity criteria.

Interpretation of results:

study cannot be used for classification

Remarks:

(positive control did not meet validity criteria)

Conclusions:

Under the study conditions, the test substance induced a significant tissue mortality and was therefore predicted to be irritant to skin. The study did not meet validity criteria, therefore the study could not be used for classification..

Executive summary:

A study was conducted to determine the in vitro skin irritation potential of the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', using Reconstructed Human Epidermis (RHE) cells, according to a method similar to OECD Guideline 439, in compliance with GLP. Three replicates of 100 µL test substance (undiluted) were applied topically for 1, 4.5 or 20 h to the model skin surface. Phosphate buffered saline (PBS) and Triton X 100 were included as negative and positive controls respectively. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. The time at which the percent viability was 50% (i.e., ET-50) was estimated for each tissue. The test substance induced a significant decrease of tissue viability with values of 13, 3 and 3% after 1, 4.5 and 20 h, respectively. The ET-50 value for test substance was determined to be less than 0.5 h. For positive control, ET-50 value was determined to be 2.9 h, which was out of domain for QC acceptance criteria (4 to 8.7 h). Therefore, the study has been considered not to fulfill validity criteria. Under the study conditions, the test substance induced a significant tissue mortality and was therefore predicted to be irritant to skin (CPT, 2003). The study did not meet validity criteria, therefore the study could not be used for classification.

Reference 4

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From May 16, 2017 to June 15, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

The reconstructed human epidermal model EpidermTM (EPI-200 MatTek Corporation)

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Justification for test system used:

The EpiDermTM skin model and assay for skin corrosion testing is endorsed by OECD TG 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

Test system
The reconstructed human epidermal model EpidermTM (EPI-200 MatTek Corporation) consists of normal human-derived epidermal keratinocytes which have been cultured to form a multi-layered highly differential model of the human epidermis. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Characterisation of the test system
MatTek’s EpiDermTM model has been extensively characterised for multiple parameters including morphology, tissue viability, skin barrier function and sterility. QC results for the specific lot of models received (Lot# 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 EpiDerm^TM tissue model was non-corrosive.

Interpretation of results:

other: CLP criteria for corrosion not met

Conclusions:

Based on the results of the read across study, the test substance is considered to be non-corrosive to the skin.

Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using Reconstructed Human Epidermis (RHE) cells, according to OECD 431 Guideline, in compliance with GLP. EpiDermTM tissues were kept overnight at 4°C. On Day 1, the tissues were pre-incubated for 1 h at 37°C, 5% CO2, 95% RH. After incubation, tissues were exposure to test (25 mg) and reference substances (25 μL sterile water as negative control and 50 μL Potassium hydroxide as positive control) in triplicates for 3 and 60 minutes. After 3 minutes and 1 h treatment, the test substance and the reference substances were rinsed off from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT on Day 2. On Day 3, final MTT assay testing and measurements were performed. Results were compared to negative control. All validity criteria for the performed test were met. After 3 minutes and 1 h treatment, the mean viability values obtained with the substance were determined to be 103.7% and 93.8%, respectively, which is well above the corrosive limits of 50 and 15% respectively. Under the study conditions, the read across substance was determined to be non-corrosive to the skin (XCellR8, 2017). Based on the results of the read across study, the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', is considered to be non-corrosive to the skin.

Reference 5

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From May 18, 2017 to June 23, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

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:

Based on the results of the read across study, the test substance is considered to be non-irritating to the skin.

Executive summary:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 06, 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:

not specified

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

not specified

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:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

32 ± 1 ºC for 120 minutes

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

ca. 52.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

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

Results

Corneal Opacity and Permeability Measurement

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

Treatment	Cornea Number	Opacity					Permeability (Optical Density)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control #	1	3	2	2	0		0.002
	2	2	3	2	0		0.002
	3	3	2	2	0		0.003
	Mean				0.0*		0.002		0.0
Positive Control #	4	0	27	30	30	30.0	1.805	1.803
	5	3	35	32	29	29.0	1.133	1.131
	6	0	27	29	29	29.0	1.043	1.041
	Mean					29.3		1.325	49.2
Test Substance	10	0	23	45	45	45.0	0.306	0.304
	11	1	37	55	54	54.0	0.088	0.086
	12	0	27	51	51	51.0	0.063	0.061
	Mean					50.0		0.150	52.3

* = Mean of the post-incubation-pre‑treatment values                           

# = Control data shared with Envigo - Shardlow study number BS88JJ

Corneal Epithelium Condition

The condition of each cornea is given in below table:

 

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control #	1	Clear	Clear
	2	Clear	Clear
	3	Clear	Clear
Positive Control #	4	Cloudy	Cloudy
	5	Cloudy	Cloudy
	6	Cloudy	Cloudy
Test Substance	10	Cloudy – with some test substance remaining	Cloudy – with some test substance remaining
	11	Cloudy – with some test substance remaining	Cloudy – with some test substance remaining
	12	Cloudy – with some test substance remaining	Cloudy – with some test substance remaining

 

The corneas treated with the test substance were cloudy post treatment and post incubation with some test substance remaining adhered to the corneas. The corneas treated with the negative control substance were clear post treatment and post incubation. The corneas treated with the positive control substance were cloudy post treatment and post incubation.

 

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Substance	52.3
Negative Control	0.0
Positive Control	49.2

 

Criteria for an Acceptable Test

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

 

Conclusion

Based on the study results, the study author concluded that no prediction of eye irritation could be made.

Interpretation of results:

other: No prediction can be made based on EU CLP criteria

Conclusions:

Under the study conditions, no prediction could be made about eye irritation potential of the test substance.

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', using Bovine Corneal Opacity and Permeability (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The undiluted test substance and reference substances were applied to the test system for 10 minutes followed by an incubation period of 120 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 test substance IVIS was 52.3, which is well below the corrosive limit of 55. Therefore no prediction can be made about eye irrtation potential of the test substance, based on EU CLP criteria. The positive control IVIS was within the range of 31.6 to 58.7 and the negative control gave opacity of ≤3.0 and permeability ≤0.077, therefore, the test was considered to have met all validity criteria. Under the study conditions, no prediction could be made about eye irritation potential of the test substance (Envigo, 2017).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: HET-CAM test

Version / remarks:

modification of that described by Kemper and Luepke

GLP compliance:

yes

Specific details on test material used for the study:

Lot no.: P-2903

Species:

chicken

Details on test animals or tissues and environmental conditions:

- Chick embryo: chorioallantoic membrane (CAM)
- Fresh, fertile, White Leghor, eggs obtained from Avian Services in Frenchtown, New Jersey
- Acclimation: 7 d at 13°C before incubation
- Incubation: ca. 37°C with a relative humidity of 60 - 70% for 10 d (in a Kuhl incubator)

Vehicle:

water

Remarks:

10% test substance in water

Controls:

yes, concurrent vehicle

Amount / concentration applied:

Test substance: 0.3 mL if liquid or 0.3 g if solid
Different concentrations: 0.5, 2.5 and 5%

Duration of treatment / exposure:

20 s

Observation period (in vivo):

Up to 5 minutes

Duration of post- treatment incubation (in vitro):

-

Number of animals or in vitro replicates:

4

Details on study design:

After washing with physiological saline, membranes were observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects.

Irritation parameter:

other:

Run / experiment:

mean scores for hyperaemia, haemorrhage, coagulation and/or thrombosis

Value:

>= 2 - <= 14.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: irritation potential: practically none to moderate

Other effects / acceptance of results:

CAM were exposed to the test substance at concentrations of 0, 0.5, 2.5 and 5% and the mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis were 1.75, 3.00, 2.00 and 14.50, respectively. The test substance was considered to have practically none to moderate irritation potential to CAM.

Conclusions:

Under the study conditions, the test substance was considered to have practically none to moderate irritation potential to eye (HET-CAM).

Executive summary:

A study was conducted to determine the in vitro eye irritation potential of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd AE5 PSE' according to Hen's Egg Test – Chorioallantoic Membrane (HET-CAM), in compliance with GLP. The CAMs were exposed to 0.3 mL or 0.3 g test substance at concentrations of 0 (vehicle only: distilled water), 0.5, 2.5 and 5% for 20 s. After washing with physiological saline, membranes were observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects. The mean scores for hyperemia, hemorrhage, coagulation and/or thrombosis were determined to be 1.75, 3.00, 2.00 and 14.50, respectively. Under the study conditions, the test substance was considered to have practically none to moderate irritation potential to eye at concentrations up to 5% (CPT, 2004).

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 27, 2017 to August 31, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

not specified

GLP compliance:

yes (incl. QA statement)

Species:

other: Epiocular™ (OCL-200) Reconstructed Human Ocular Epithelium

Strain:

other: Stratified human keratinocytes (4F1188) in a three-dimensional structure

Details on test animals or tissues and environmental conditions:

Description of the 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.

Justification for selection of the test system
The EpiOcular™ Eye Irritation Test (EIT), using the MatTek EpiOcularTM tissue model OCL-200, was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcularTM EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose, as an alternative to Draize Rabbit Eye Test with excellent correlation -In vivo to In vitro test results.

Characterisation of the test system
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:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50µL neat test substance or reference substance

Duration of treatment / exposure:

30 minutes ± 2 minutes

Duration of post- treatment incubation (in vitro):

12 ± 2 minutes’ post-treatment immersion, and 2 hours ± 15 minutes’ post-treatment incubation, prior to the MTT endpoint

Number of animals or in vitro replicates:

Three tissues per condition (n=3)

Irritation parameter:

other: Percentage (%) of viability (relative to negative control))

Remarks:

Test substance

Run / experiment:

Mean value of three runs

Value:

ca. 2.75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results

Prior to the study, the required compatibility checks confirmed that the test substance did not interfere with MTT or solvent.

Table 1: Viability measurements after 30 minutes (± 2 min) of application and 2h (± 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	2.223	2.251	2.052	2.080	2.066	105.186
	Tissue 2	2.027	2.058	1.856	1.887	1.871	95.281
	Tissue 3	2.116	2.136	1.945	1.965	1.955	99.533
PC	Tissue 1	0.949	0.95	0.778	0.779	0.778	39.620
	Tissue 2	0.817	0.826	0.646	0.655	0.650	33.101
	Tissue 3	0.783	0.8	0.612	0.629	0.620	31.574
TA2	Tissue 1	0.287	0.257	0.116	0.086	0.101	5.118
	Tissue 2	0.193	0.189	0.022	0.018	0.020	0.993
	Tissue 3	0.231	0.196	0.060	0.025	0.042	2.139

NC: negative control (sterile H₂O), PC: Positive control (neat Methyl Acetate), TA2: Test substance

 

Table 2: Mean and SD of viability measurements and of viability percentages after 30 minutes (± 2 min) of application and 2h (± 15 min) post-incubation.

Name	Code	Mean of OD	SD of OD	Mean of viability (%)	SD of viability (%)	CV %	Classification
Sterile water	NC	1.964	0.098	100.000	4.969	4.969	Non-Irritant
Methyl Acetate	PC	0.683	0.084	34.765	4.273	12.292	Irritant
Test substance	TA2	0.054	0.042	2.750	2.129	77.430	Irritant

Evaluation of the Results

Results were checked against the following 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.	1.964	PASS
Acceptance criterion 2	The mean of the positive control relative percentage viability is below 50% of negative control viability after 30 minutes exposure.	34.765	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: 4.969 PC: 4.273 Test Substance: 2.129	PASS

 

All acceptance criteria were met during the study.

 

Interpretation of Results following Prediction Model

 

A test substance is considered to be irritant to the eye (i.e. would require labelling as either GHS 1 or 2) if the eye model viability after exposure and post-treatment incubation is ≤60%.

 

A test substance is considered as a non-irritant to the eye (i.e. would not require a warning label in the European chemical classification systems) if the eye model viability after exposure and post-treatment incubation is >60%.

 

The current assay is not intended to differentiate between GHS class 1 and GHS class 2 or R36 and R41 (degree of stromal damage). The percentage of viability obtained with test substance was 2.750%, therefore it was considered as Irritant to the eye.

Interpretation of results:

other: Category 1 (irreversible effects on the eye) based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was classified as irritant to the human eye.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 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. After pre-wetting the tissues with 20µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, test system was exposed to 50µL of the test substance or reference substances (negative control: sterile water; positive control: methyl acetate) for 30 minutes ± 2 minutes, followed by a 12 ± 2 minutes post-treatment immersion, and 2 h ± 15 minutes post-treatment incubation. After post-treatment incubation period, MTT test was performed with readings at 570 nm without reference filter, and percentage viability value for EpiOcularTM models exposed to the test substance relative to the negative control was calculated. The percentage viability obtained for negative control, positive control and test substance were calculated be 100%, 34.77% and  2.750% respectively. The test had passed all validity criterias.Since the percentage viability of the test substance was much below the threshold indicating no irritation potential, the test substance was classified as irritant to the human eye by the study authors (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.

Reference 4

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, is considered as inducing serious eye damage and classified as Category 1 (irreversible effects on the eye) based on EU CLP criteria.

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the the 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- C18-unsatd. PSE and C18-unsatd. AE5 PSE'.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

Study 1 – Skin corrosion:

An invitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd. AE5 PSE', using Reconstructed Human Epidermis (RHE) cells, according to a method similar to OECD Guideline 431, in compliance with GLP. Three replicates of 50 µL test substance (undiluted) were applied topically for 3 minutes or 1 h to the model skin surface. Distilled water and potassium hydroxide were used as negative and positive controls respectively. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. The time at which 50% percent viability was acheived (ET-50) was estimated for each tissue. The test substance did not induce a significant decrease of tissue viability with values of 78 and 91% after 3 min and 1 h, respectively. The positive control induced a significant decrease of tissue viability with values of 29 and 17% after 3 min and 1 h, respectively. Therefore, the study met the validity criteria. Under the study conditions, the test substance (at pH 6.20) was predicted to be non-corrosive to skin (CPT, 2004).

Study 2 – Skin corrosion:

An in vitro study was conducted to determine the corrosive potential and to designate the Packing Group classification of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd. AE5 PSE' using the Corrositex test method. This test predicts the in vivo corrosive potential of a chemical compound or mixture by using an endpoint, i.e. the time it takes for the chemical to permeate through or destroy a synthetic biobarrier. When a chemical has passed through this biobarrier, a visual change is produced in a proprietary chemical detection system (CDS). The test is performed in 3 steps. A qualification test is done to insure that the test substance sample and the CDS reagent are compatible (observation of any changes). The appropriate indicator solutions are then selected in order to permit categorization of the sample as either a Category 1 or 2 material. Finally, the test substance is applied on the biobarrier. The changes and the time at which they appear, are recorded following visual observation. The test substance is then classified according to the occurence of the changes (Packing I (0 to 3 min), II (3 to 60 min), III (30 to 240 min) or non-corrosive (> 60 min), depending on the category). Four replicates were analysed in this experiment. The test substance sample was compatible with the Corrositex system and was classified as a Category 2 material. The results for the 4 replicates were highly reproducible and a mean time of > 60 minutes was required to destroy the synthetic barrier. Under the study conditions, the test substance was considered non-corrosive (IVI, 2005).

Study 3 – Skin irritation:

An in vitro study was conducted to determine the skin irritation potential of the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', using Reconstructed Human Epidermis (RHE) cells, according to a method similar to OECD Guideline 439, in compliance with GLP. Three replicates of 100 µL test substance (undiluted) were applied topically for 1, 4.5 or 20 h to the model skin surface. Phosphate buffered saline (PBS) and Triton X 100 were included as negative and positive controls respectively. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. The time at which the percent viability was 50% (i.e., ET-50) was estimated for each tissue. The test substance induced a significant decrease of tissue viability with values of 13, 3 and 3% after 1, 4.5 and 20 h, respectively. The ET-50 value for test substance was determined to be less than 0.5 h. For positive control, ET-50 value was determined to be 2.9 h, which was out of domain for QC acceptance criteria (4 to 8.7 h). Therefore, the study has been considered not to fulfill validity criteria. Under the study conditions, the test substance induced a significant tissue mortality and was therefore predicted to be irritant to skin (CPT, 2003). The study did not meet validity criteria, therefore the study could not be used for classification.

Study 4 - Skin corrosion - read across study:

An in vitro study was conducted to determine the skin corrosion potential of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using Reconstructed Human Epidermis (RHE) cells, according to OECD 431 Guideline, in compliance with GLP. EpiDermTM tissues were kept overnight at 4°C. On Day 1, the tissues were pre-incubated for 1 h at 37°C, 5% CO2, 95% RH. After incubation, tissues were exposure to test (25 mg) and reference substances (25 μL sterile water as negative control and 50 μL Potassium hydroxide as positive control) in triplicates for 3 and 60 minutes. After 3 minutes and 1 h treatment, the test substance and the reference substances were rinsed off from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT on Day 2. On Day 3, final MTT assay testing and measurements were performed. Results were compared to negative control. All validity criteria for the performed test were met. After 3 minutes and 1 h treatment, the mean viability values obtained with the substance were determined to be 103.7% and 93.8%, respectively, which is well above the corrosive limits of 50 and 15% respectively. Under the study conditions, the read across substance was determined to be non-corrosive to the skin (XCellR8, 2017). Based on the results of the read across study, the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', is considered to be non-corrosive to the skin.

Study 5 - Skin irritation - read across study:

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

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

Overall, based on the degree of ethoxylation trend noted in the HERA report (which likely explains the conclusion observed in the read across study), together with the absence of corrosive evidence in available in vitro studies indicates that the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', can be considered to be irritating to skin (as worst case).

Eye: 

Study 1 – Eye irritation:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd AE5 PSE' according to Hen's Egg Test – Chorioallantoic Membrane (HET-CAM), in compliance with GLP. The CAMs were exposed to 0.3 mL or 0.3 g test substance at concentrations of 0 (vehicle only: distilled water), 0.5, 2.5 and 5% for 20 s. After washing with physiological saline, membranes were observed at 0.5, 2 and 5 minutes post-application. The reactions of the CAM, the blood vessels, including the capillaries and the albumin were examined and scored for irritant effects. The mean scores for hyperaemia, haemorrhage, coagulation and/or thrombosis were determined to be 1.75, 3.00, 2.00 and 14.50, respectively. Under the study conditions, the test substance was considered to have practically none to moderate irritation potential to eye at concentrations up to 5% (CPT, 2004).

 

Study 2 – Eye irritation:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 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. After pre-wetting the tissues with 20µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, test system was exposed to 50µL of the test substance or reference substances(negative control: sterile water; positive control: methyl acetate)for 30 minutes ± 2 minutes, followed by a 12 ± 2 minutes post-treatment immersion, and 2 h ± 15 minutes post-treatment incubation. After post-treatment incubation period, MTT test was performed with readings at 570 nm without reference filter, and percentage viability value for EpiOcularTM models exposed to the test substance relative to the negative control was calculated. The percentage viability obtained for negative control, positive control and test substance were calculated be 100%, 34.77% and  2.750% respectively. The test had passed all validity criterias.Since the percentage viability of the test substance was much below the threshold indicating no irritation potential, the test substance was classified as irritant to the human eye by the study authors (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 – Eye corrosion:

An in vitro study was conducted to determine the eye irritation potential of the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', using Bovine Corneal Opacity and Permeability (BCOP) method, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. The undiluted test substance and reference substances were applied to the test system for 10 minutes followed by an incubation period of 120 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 test substance IVIS was 52.3, which is well below the corrosive limit of 55. Therefore no prediction can be made about eye irritation potential of the test substance, based on EU CLP criteria. The positive control IVIS was within the range of 31.6 to 58.7 and the negative control gave opacity of ≤3.0 and permeability ≤0.077, therefore, the test was considered to have met all validity criteria. Under the study conditions, no prediction could be made about eye irritation potential of the test substance (Envigo, 2017).

Study 4 - Eye corrosion - read across study

An in vitro 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 criteria. 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- C18-unsatd. PSE and C18-unsatd. AE5 PSE'.

Further, as per a HERA 2009 review report, no relationship could be established between the chemical structures of the tested AEs and their eye irritation responses (HERA, 2009). Therefore, in absence of clear conclusions in the in vitro tests with the test substance and based on the results of thein vitro read across study, the test substance, ‘mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE' can be considered to be corrosive to eyes (in the worst case).

Justification for classification or non-classification

Skin irritation:

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

Eye irritation:

Based on the available weight of evidence from in vitro studies with the test and read across substances, the test substance, ‘mono- and di- C18-unsatd.. PSE and C18-unsatd.. AE5 PSE’, warrants an ‘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.