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Description of key information

Based on the results of the in vitro eye and skin irritation studies, the test substance, 'di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS', is considered to be non-irritating to the skin and irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 20, 2017 to May 13, 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:
Optical Density (OD) values obtained with blanks were higher than 0.1 (0.184) causing a deviation from Acceptance Criteria. However, this is not considered to be an issue in the interpretation of this study data.
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed human epidermal model EpiDermTM
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:
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.

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 mg of neat test substance.
Duration of treatment / exposure:
60 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, 95% RH).
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
Three tissues per condition (n=3).
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes exposure
Value:
96.6
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
All validity criteria for the test were met:
- Criteria: the mean OD570 of the negative control (treated with DPBS) tissues is ≥ 0.8 and ≤ 2.8.
Result for the test: 1.445

- The mean of the positive control relative percentage viability must be ≤ 20 % of the mean of the negative controls.
Result for the test: 2.7 %

- The standard deviation of OD values for triplicate skin models in each expeimental condition must be < 18 %.
Results for the test:
NC: 6.02 %
PC: 1.11 %
Test substance: 1.62 %

Optical Density (OD) values obtained with blanks were higher than 0.1 (0.184) causing a deviation from acceptance criteria 4. However, the spectrophotometer was fully validated and had passed all required tests. The OD values for blanks observed in this study are consistent with historical data using this spectrophotometer in the XCellR8 laboratory and meet our current internal acceptance criteria of blank OD values <0.194 (mean of XCellR8 historical data, based on blanks obtained during 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:
other: CLP criteria not met
Conclusions:
Under the study conditions, the percentage viability for the test substance was established at 96.6% in EpiDerm™ model, therefore 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 test substance, ‘di-C16-18 -satd. and C18-24-unsatd. AAEMIM-MS (active: 100%)' using Reconstructed Human Epidermis (RHE) cells, according to OECD 439 Guideline, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, 95% RH. On Day 1, the tissues in triplicate were exposed to nominal 25 mg of test substance and 30 µL reference substances, applied topically for 60 ±1 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, 95% RH), followed by rinsing steps and a 42 ± 4 h post-dose incubation at 37°C, 5% CO2, 95%RH). On Day 2, the medium was changed and on Day 3, MTT viability test with readings at 570 nm without reference filter was performed. 30 µL of DPBS and 5% SDS were used as negative control and positive control, respectively. Viability of the tissues was assessed in MTT test and compared to the negative control. The percentage of viability obtained with the test substance was 96.6%, which is well above the irritant limit of 50%. The study met all the validity criteria. Under the study conditions, the test substance was determined be non-irritating to the skin (XCellR8, 2017).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From August 31, 2017 to August 31, 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)
Version / remarks:
updated 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
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):
2 h
Number of animals or in vitro replicates:
Triplicate
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 substances.

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

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

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

Histopathology: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
Irritation parameter:
in vitro irritation score
Run / experiment:
10 minutes
Value:
4.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Inconclusive

Corneal epithelium condition: The corneas treated with the test substance were clear post treatment and partly cloudy post incubation. 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.

Individual and mean corneal opacity and permeability measurements:

Treatment Cornea Number Opacity Permeability (OD) In Vitro Irritancy Score
Pre-Treatment Post-Treatment Post Incubation Post-Incubation - Pre-Treatment Corrected Value   Corrected Value
Negative Control 1 3 3 4 1   0.007    
2 3 2 2 0   0.011    
3 4 2 2 0   0.007    
        0.3*   0.008♦   0.5
Positive Control 4 6 40 37 31 30.7 0.794 0.786  
5 3 31 33 30 29.7 1.96 1.952  
6 2 24 26 24 23.7 1.885 1.877  
          28•   1.538• 51.1
Test substance 10 3 6 6 3 2.7 0.099 0.091  
11 3 4 9 6 5.7 0.073 0.065  
12 1 3 4 3 2.7 0.014 0.006  
          3.7•   0.054• 4.5

OD = Optical density * = Mean of the post-incubation − pre-treatment values ♦ = Mean permeability • = Mean corrected value

Corneal epithelium condition post treatment and post incubation

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 Clear Partly Cloudy
11 Clear Partly Cloudy
12 Clear Partly Cloudy

Results:

Treatment In Vitro Irritancy Score
Test substance 4.5
Negative control 0.5
Positive control 51.1
Interpretation of results:
other: Inconclusive for classification
Conclusions:
Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (IVIS score – 4.5).
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS' (active: 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 (750 µL), negative control (Sodium chloride 0.9% w/v) and positive control (Ethanol) substances at 32 ± 1ºC for 120 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 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control resulted in opacity of <3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 4.5, which is well below the corrosive limit of 55 and is slightly above the non-corrosive limit of 3; therefore no predictions could be made. 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:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 21, 2018 to March 01, 2018
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:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: EpiOcularTM tissue model (OCL-200-MatTek Corporation)
Details on test animals or tissues and environmental conditions:
Test system:
The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

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. QC results for the specific lot of models received (Lot# 23787) were checked in-house for MatTek acceptance ranges with the following outcome:

- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 0.3% Triton X-100) where ET50 is the time taken for 0.3% Triton X-100 to reduce the viability of the skin model to 50% relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS
Vehicle:
other: Phosphate Buffered Saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
20 µl of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) plus 50 mg of test substance
Duration of treatment / exposure:
Pre-wetting tissues with PBS for 30 ± 2 min and application of test substance for 6h ± 15 minutes
Duration of post- treatment incubation (in vitro):
25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation
Number of animals or in vitro replicates:
3 replicates for the test substance, positive and negative control.
Details on study design:
Preliminary test:
The test substance was first checked for its potential for MTT interference and solvent interference (water and isopropanol).

Main test overview:
Day 0: On the day of receipt, EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH.
Day 1: Exposure to and removal of test and reference substances (nominal 50 mg of test substance or 50µl of reference substance for 6 h± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation).
Day 2: End of MTT viability test, readings at 570 nm without reference filter.
Irritation parameter:
other: % viability
Run / experiment:
1 h exposure
Value:
12.677
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- Prior to the study, the required compatibility checks (as per SOP L0069) confirmed that the test substance did not interfere with MTT or solvent.
- No prediction can be made for test substance as the viability was below 60%.

All acceptance criteria were met during the study:
- The mean OD570 of the negative control (treated with sterile water) tissues is > 0.8 and < 2.5.
Result: 1.902
- The mean of the positive control relative percentage viability is below 50% of negative control viability after 6h exposure.
Result: 2.882
- 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).
Results:
NC: 4.745
PC: 2.569
Test substance: 3.279

Viability measurements after 6h (± 15 min) of application and 18h (± 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 1.949 1.934 1.84 1.825 1.832 96.312
Tissue 2 1.979 1.981 1.87 1.872 1.871 98.335
Tissue 3 2.147 2.08 2.038 1.971 2.004 105.353
PC Tissue 1 0.14 0.138 0.031 0.029 0.03 1.559
Tissue 2 0.219 0.222 0.11 0.113 0.111 5.844
Tissue 3 0.132 0.134 0.023 0.025 0.024 1.244
Teest substance Tissue 1 0.276 0.283 0.167 0.174 0.17 8.945
Tissue 2 0.37 0.381 0.261 0.272 0.266 13.992
Tissue 3 0.397 0.396 0.288 0.287 0.287 15.095

NC: negative control (sterile H2O),

PC: Positive control (neat Methyl Acetate),

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

Name Code Mean of OD SD of OD CV % Classification
Sterile water NC 1.902 0.09 100 4.75 4.745 No-Category
Methyl Acetate PC 0.055 0.049 2.882 2.569 89.14 No Prediction
Test substance TA1 0.241 0.062 12.677 3.279 25.865 No Prediction
Interpretation of results:
study cannot be used for classification
Conclusions:
Under the study conditions, the percentage viability obtained was 12.677% and therefore, the test substance was classified as irritant to the human eye. However, based on the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage).
Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘di-C16-18-satd. and C18-24 unsatd. AAEMIM-MS' (active: 100%), 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 50 mg of the test substance or reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation. 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%, 12.677% and 2.882% 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, 2018). However, based on the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage), hence, the classification for the test substance is considered to be inconclusive.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin:

An in vitro study was conducted to determine the skin irritation potential of the test substance, ‘di-C16-18 -satd. and C18-24-unsatd. AAEMIM-MS (active: 100%)' using Reconstructed Human Epidermis (RHE) cells, according to OECD 439 Guideline, in compliance with GLP. EpiDermTM tissues were pre-incubated overnight at 37°C, 5% CO2, 95% RH. On Day 1, the tissues in triplicate were exposed to nominal 25 mg of test substance and 30 µL reference substances, applied topically for 60 ±1 minutes (25 minutes at room temperature and 35 minutes at 37°C, 5% CO2, 95% RH), followed by rinsing steps and a 42 ± 4 h post-dose incubation at 37°C, 5% CO2, 95%RH). On Day 2, the medium was changed and on Day 3, MTT viability test with readings at 570 nm without reference filter was performed. 30 µL of DPBS and 5% SDS were used as negative control and positive control, respectively. Viability of the tissues was assessed in MTT test and compared to the negative control. The percentage of viability obtained with the test substance was 96.6%, which is well above the irritant limit of 50%. The study met all the validity criteria. Under the study conditions, the test substance was determined be non-irritating to the skin (XCellR8, 2017).

Eye:

Study 1:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘di-C16-18-satd. and C18-24 unsatd. AAEMIM-MS' (active: 100%), 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 50 mg of the test substance or reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation. 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%, 12.677% and 2.882% 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, 2018). 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 2:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS' (active: 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 (750 µL), negative control (Sodium chloride 0.9% w/v) and positive control (Ethanol) substances at 32 ± 1ºC for 120 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 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control resulted in opacity of <3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied. The test substance IVIS score obtained was 4.5, which is well below the corrosive limit of 55 and is slightly above the non-corrosive limit of 3; therefore no predictions could be made. Under the study conditions, no prediction of eye irritation could be made for the test substance (Envigo, 2018).

Based on the two in vitro eye irritation studies were conducted with test substance, no clear conclusions could be drawn as per the Guidelines. However, given the IVIS score from BCOP study, which was closer to the non-corrosive limit and the percentage viability from the RhCE test, which was quiet low, together with the absence of skin irritation potential (as assessed based on in vitro RHE OECD 439 Guideline study), indicate that a corrosive potential can be ruled out and the test substance, 'di-C16-18 satd. and C18-24-unsatd. AAEMIM-MS' can be considered to be more likely to be irritating to the eyes (in a worst case).

Justification for classification or non-classification

Skin irritation:

Based on the results of the in vitro assay, the test substance, 'di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS', does not warrant classification for skin irritation, according to the EU CLP criteria (Regulation 1272/2008/EC).

 

Eye irritation:

Based on the results of in vitro assays and as a conservative approach, the test substance, 'di-C16-18 -satd. and C18-24-unsatd. AAEMIM-MS', is considered to warrant 'Eye Irrit. 2: H319 - causes serious eye irritation’ classification according to the EU CLP criteria (Regulation 1272/2008/EC).