Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From January 01, 1986 to February 18, 1986
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 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.6 (Skin Sensitization)
Deviations:
no
GLP compliance:
no
Species:
rabbit
Strain:
other: Kleinrusse Chbb
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HM/Fa. Thomae, Germany
- Average weight at study initiation: 2,192 g


Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
Undiluted
Duration of treatment / exposure:
4 h
Observation period:
21 d
Number of animals:
5
Details on study design:
24 h before test start, the right flank of the rabbits was shaved and the skin inspected for intactness. Only rabbits with intact skin were used for the study. 0.5 mL of the undiluted test substance were applied to a 2.5 x 2.5 cm patch and placed onto the shaved skin, then securely fastened with a larger plastic sheet to provide an occlusive dressing. After 4 h, the dressing was removed and the skin was scored for reddening (erythema) and swelling (oedema) after 1, 24, 48 and 72 h, then 7, 10, 14, 17 and 21 d according to the Draize system.
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
3.96
Max. score:
4
Reversibility:
fully reversible within: 14 d
Remarks on result:
positive indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
3.12
Max. score:
4
Reversibility:
fully reversible within: 14 d
Remarks on result:
probability of weak irritation
Irritant / corrosive response data:
Light to moderate erythema and light oedema were observed on the treated skin after removal of the patch. The reactions intensified until the 72 h observation time point when strong erythema and moderate to strong oedema, as well as eschar formation were seen. No or only traces of the eschar were left in 1/5 and 4/5 animals, respectively, after 21 d. The acute reactions were no longer visible after 14 d.

Results table:

Product Animal number Hours after the plaster removal Days after the plaster removal
immediate 1 24 48 72 7 10 14 17 21
E O E O E O E O E O E O E O E O E O E O N S
ke 421 left side 339 3 2 4 2 4 4 4 4 4 4 4 2 4 2 4 0 4 0 4 0 N S Na
342 4 2 4 2 4 4 4 4 4 4 4 2 4 2 4 0 4 0 4 0 N S
352 4 2 4 2 4 4 4 4 4 4 4 2 4 2 4 2 4 2 4 0 N S
355 4 2 4 2 4 4 4 4 4 4 4 2 4 3 4 2 4 1 4 0 N S
369 4 2 4 2 4 4 4 3 4 3 4 2 4 2 1 0 0 0 0 0 S Na
X 5.8 6 8 7.8 7.8 6 6.2 4.2 3.8 3.2
X% 72.5 75 100 97.5 97.5 75 77.5 52.5 47.5 40
Right side 339 1 0 2 0 3 2 4 3 4 3 3 2 3 2 1 0 0 0 0 0
342 1 1 2 1 3 2 3 3 4 3 4 2 3 2 1 0 0 0 0 0 Sx
352 1 1 1 1 3 1 4 3 4 3 2 2 2 2 0 0 0 0 0 0 Sx
355 2 1 2 1 3 2 3 3 4 3 2 1 1 0 0 0 0 0 0 0 Sx
369 2 1 1 1 3 2 4 3 4 4 1 0 1 0 0 0 0 0 0 0 Sx
X 2.2 2.4 4.8 6.6 7.2 3.8 3.2 0.4 0 0
X% 27.5 30 60 82.5 90 47.5 40 5 0 0

E = Erythema

O = Odema

N = Necrotic skin change

S = Scab

Sx = Punctiform scraps

Na = Scar

Interpretation of results:
other: Category 2 (irritant) based on CLP criteria
Conclusions:
Under the test conditions, read across substance, was considered to be irritating to rabbit skin.
Executive summary:

An in vivo study was conducted to assess the skin irritation potential of read across substance, C12-18 and C18 -unsatd. DEA, to rabbit skin according to OECD Guideline 404. In this study, shaved skin of 5 rabbits was exposed to the undiluted test substance using occlusive patches for 4 h. The skin was then observed for effects for 21 d after patch removal andscored according to the Draize system. Exposure to undiluted test substance caused light to moderate erythema and light oedema immediately after removal of the patch. The reactions intensified until the 72 h observation time point when strong erythema and moderate to strong oedema, as well as eschar formation were seen. No or only traces of the eschar were left in 1/5 and 4/5 animals, respectively, after 21 d. The acute reactions were no longer visible after 14 d. Under the test conditions, read across substance was considered to be irritating to rabbit skin (Kästner, 1986). Based on the results of the read across study, the test substance, C10 -12 and C18 -unsatd. DEA, is also considered to be irritating to rabbit skin.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 16, 2017 to June 15, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Supplier batch/lot No.: 0001163765; Purity: 100 %
Test system:
human skin model
Source species:
other: Reconstructed human epidermal model EpidermTM (EPI-200 MatTek Corporation)
Cell type:
other: 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:
Description of the 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:
50 μl of neat test substance
Duration of treatment / exposure:
3 and 60 minutes
Number of replicates:
Triplicates for the test substance, negative and positive control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
112.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
83.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non corrosive
Other effects / acceptance of results:
All validity criteria were met:

- The mean OD570 of the negative control tissues must be ≥0.8.
Results: 1.651 after 3 min, 1.869 after 1h

- The mean of the positive control relative percentage viability, after 1 hour exposure must be <15 % of the mean of the negative control.
Result: 3.0 %

- 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 %).
Results:
NC: 5.6 % after 3 min, 5.6 % after 1h
PC: 22.8 % after 3 min, 15.0 % after 1h
TA1: 6.1 % after 3 min, 15.4 % after 1h

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 1.751 1.732 1.742 1.651 105.5 100 5.6 5.6
2 1.668 1.639 1.654 100.2
3 1.6 1.513 1.557 94.3
Test substance 1 1.99 1.97 1.98 1.851 120 112.1 5.6 6.1
2 1.804 1.807 1.806 109.4
3 1.793 1.742 1.768 107.1
PC 1 0.548 0.574 0.561 0.398 34 24.1 10.1 42*
2 0.223 0.231 0.227 13.8*
3 0.403 0.407 0.405 24.5

NC: negative control (H2O),

PC: Positive control (KOH 8N)

*CV value of the PC is outlier above 30%

Cell viability measurements after 3 minutes of application (corrected)

Name Tissue n° 3 min endpoint
Aliq. 1 Aliq. 2 mean OD Mean Viability Mean SD CV
% % % %
NC 1 1.751 1.732 1.742 1.651 105.5 100 5.6 5.6
2 1.668 1.639 1.654 100.2
3 1.6 1.513 1.557 94.3
Test substance 1 1.99 1.97 1.98 1.851 120 112.1 6.9 6.1
2 1.804 1.807 1.806 109.4
3 1.793 1.742 1.768 107.1
PC 1 0.548 0.574 0.561 0.483 34 29.3 6.7 22.8
2 0.223 0.231   *
3 0.403 0.407 0.405 24.5

NC: negative control (H2O),

PC: Positive control (KOH 8N)

*CV value of the PC is outlier above 30%

Cell viability measurements after 1 h of application results

Name Tissue n° 1 h endpoint
Aliq. 1 Aliq. 2 mean OD Mean Viability Mean SD CV
% % % %
NC 1 1.904 1.879 1.891 1.869 101.2 100 5.6 5.6
2 1.95 1.974 1.962 105
3 1.79 1.719 1.754 93.9
Test substance 1 1.446 1.454 1.45 1.869 77.6 83.5 12.8 1.446
2 1.835 1.836 1.835 98.2 1.835
3 1.434 1.357 1.395 74.6 1.434
PC 1 0.037 0.06 0.048 0.057 2.6 3 0.5 15
2 0.052 0.06 0.057 3.1
3 0.059 0.072 0.065 3.5

NC: negative control (H2O),

PC: Positive control (KOH 8N)

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

    3 min     1 h  
Mean of viability [%] SD of viability CV(%) Mean of viability [%] SD of viability CV(%)
NC 100 5.6 5.6 100 5.6 5.6
Test substance 112.1 6.9 6.1 83.5 12.8 15.4
PC 29.3 6.7 22.8 3 0.5 15
Interpretation of results:
other: CLP criteria not met
Remarks:
(non-corrosive)
Conclusions:
Under the study conditions the test substance is considered as non-corrosive to skin.
Executive summary:

An in vitro study was conducted to determine the skin corrosion potential of the test substance, C10-12 and C18-unsatd. DEA, using Reconstructed Human Epidermis (RHE) test Method, according to OECD 431 Guideline, in compliance with GLP. One valid experiment was performed. Three tissues of the human skin model EpiDermTM were treated with the test substance for 3 minutes and 1 h, respectively. 50 µL of the test substance was topically applied to each tissue. Demineralised water was used as negative control, and KOH as positive control. After treatment, the test substance or the control substance was rinsed off from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT. After treatment with the negative control, the absorbance values were within the required acceptability criterion, showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals. After 3 minutes treatment, the mean viability values obtained with the test substance was decreased to 112.1% compared to the negative control. This value is well above the threshold indicating corrosivity (50%). After 1 h treatment the mean viability value was reduced to 83.5%. This value is well above the threshold for corrosivity (15%) as well. Under the study conditions the test substance was considered as non-corrosive to skin (XCELLR8, 2017).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 05, 2017 to December 05, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
updated 26 July 2013
Deviations:
yes
Remarks:
This deviation was considered to have not affected the integrity or validity of the study.
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
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):
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:
62.7
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

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 2 4 1   0.003    
2 3 3 7 4   0.005    
3 4 5 8 4   0.035    
        0.3*   0.014♦   3.2
Positive Control 4 4 30 30 26 23.0 0.340 0.326  
5 4 37 33 29 26.0 0.466 0.452  
6 3 33 28 25 22.0 0.448 0.434  
          23.7•   0.404• 29.7
Test substance 10 2 29 71 69 66 0.561 0.547  
11 1 43 66 65 65 0.076 0.062  
12 1 48 53 52 52 0.155 0.141  
          59.0•   0.250• 62.7

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  Cloudy Cloudy
11  Cloudy Cloudy
12  Cloudy Cloudy

Results:

Treatment In Vitro Irritancy Score
Test substance 62.7
Negative control 3.2
Positive control 29.7
Interpretation of results:
other: Category 1 (irreversible effects on the eye) based on CLP criteria
Conclusions:
Under the study conditions, the test substance was found to be causes serious eye damage on bovine corneal opacity and permeability test (IVIS score – 62.7).
Executive summary:

An in vitro study was conducted to determine the eye damage potential of the test substance, C10-12 and C18-unsatd. DEA, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. In this study, eye damage of the test substance was tested through topical application for 10 minutes, followed by an incubation period of 120 minutes. The test substance was applied as is (750 µL) directly on top of the freshly isolated bovine cornea sample. Negative and positive control items 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 negative control responses for opacity and permeability was less than the upper limits of the laboratory historical range, indicating that the negative control did not induce irritancy on the corneas. The mean IVIS of the positive control (Ethanol) was 29.7, which was marginally lower than the criteria range set for an acceptable test. As the score was only marginally lower, it was decided that the result was acceptable as the positive control group still provided its intended function, which was to show the sensitivity of the test system to a known ocular irritant. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The IVIS score of the test substance was determined to be 62.7 after 10 minutes of treatment, which is above the IVIS threshold of 55, indicating Category 1 conclusion. Under the study conditions, the test substance was concluded to cause serious eye damage (Envigo, 2018).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 25, 2017 to June 22, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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. certificate)
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:
physiological 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 µl of test substance
Duration of treatment / exposure:
30 minutes ± 2 min followed by a 12 ± 2 minutes post-treatment immersion.
Duration of post- treatment incubation (in vitro):
2 hours ± 15 min
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.
Day 1: Exposure to and removal of test and reference substances (50 µl of test substance or reference substances for 30 minutes ± 2 minutes, followed by a 12 ± 2 minutes post-treatment immersion, and 2 hours ± 15 minutes’ post-treatment incubation). Start of MTT viability test.
Day 2: End of MTT viability test, readings at 570 nm without reference filter
Irritation parameter:
other: % viability
Run / experiment:
30 minutes
Value:
22.497
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 item did not interfere with MTT or solvent.
- The test substance reduced the viability to below 60% after 30 minutes of application and should be considered as irritant to the eye.

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: 1.793
- The mean of the positive control relative percentage viability is below 50 % of negative control viability after 30 minutes exposure.
Result: 37.306
- 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: 3.712
PC: 8.501
TA1: 3.704

Viability measurements after 30 minutes (± 2 min) of application and 2 h (± 15 min) post-incubation of test and reference substances.

Condition Tissue # Raw data Blank corrected data Mean OD % of viability
aliquot 1 aliquot 2 aliquot 1 aliquot 2
NC Tissue 1 2.002 2.023 1.822 1.843 1.833 102.213
Tissue 2 1.989 2.031 1.809 1.851 1.83 102.073
Tissue 3 1.894 1.898 1.714 1.718 1.716 95.714
PC Tissue 1 1.005 1.032 0.825 0.852 0.839 46.77
Tissue 2 0.713 0.734 0.533 0.554 0.544 30.315
Tissue 3 0.794 0.815 0.614 0.635 0.625 34.833
Test substance Tissue 1 0.502 0.521 0.322 0.341 0.332 18.49
Tissue 2 0.586 0.606 0.406 0.426 0.416 23.203
Tissue 3 0.632 0.653 0.452 0.473 0.463 25.797

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 1.793 0.067 100 3.712 3.712 Non-Irritant
Methyl Acetate PC 0.669 0.152 37.306 8.501 22.788 Irritant
Test substance 0.403 0.066 22.497 3.704 16.466 Irritant
Interpretation of results:
study cannot be used for classification
Remarks:
Inconclusive results
Conclusions:
Under the study conditions, the percentage viability obtained was 22.497 % 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, C10-12 and C18 -unsatd. DEA, using Reconstructed human Cornea-like Epithelium (RhCE) test method, according to OECD 492 Guideline, in compliance with GLP. Three tissues of the EpiOcularTM tissue model were treated with the test substance, positive or negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) prior to topical application of approximately 50 µL of the neat test substance. Sterile water was used as negative control and methyl acetate as positive control. After 30 minutes exposure on the surface of EpiOcularTM reconstructed ocular epithelium, followed by a 12 post-treatment immersion and 2 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The percentage viability for the test substance was determined to be 22.497%, which is well below the threshold indicating irritation potential. Therefore, the study authors concluded the test substance as irritant to the human eye under the study conditions (XCELLR8, 2017). However, based on the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage), hence, the classification for the test substance is considered to be inconclusive.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Study 1: An in vitro study was conducted to determine the skin corrosion potential of the test substance, C10-12 and C18-unsatd. DEA, using Reconstructed Human Epidermis (RHE) test Method, according to OECD 431 Guideline, in compliance with GLP. One valid experiment was performed. Three tissues of the human skin model EpiDermTM were treated with the test substance for 3 minutes and 1 h, respectively. 50 µL of the test substance was topically applied to each tissue. Demineralised water was used as negative control, and KOH as positive control. After treatment, the test substance or the control substance was rinsed off from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT. After treatment with the negative control, the absorbance values were within the required acceptability criterion, showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals. After 3 minutes treatment, the mean viability values obtained with the test substance was decreased to 112.1% compared to the negative control. This value is well above the threshold indicating corrosivity (50%). After 1 h treatment the mean viability value was reduced to 83.5%. This value is well above the threshold for corrosivity (15%) as well. Under the study conditions the test substance was considered as non-corrosive to skin (XCELLR8, 2017).

Study 2: An in vivo study was conducted to assess the skin irritation potential of read across substance, C12-18 and C18 -unsatd. DEA, to rabbit skin according to OECD Guideline 404. In this study, shaved skin of 5 rabbits was exposed to the undiluted test substance using occlusive patches for 4 h. The skin was then observed for effects for 21 d after patch removal andscored according to the Draize system. Exposure to undiluted test substance caused light to moderate erythema and light oedema immediately after removal of the patch. The reactions intensified until the 72 h observation time point when strong erythema and moderate to strong oedema, as well as eschar formation were seen. No or only traces of the eschar were left in 1/5 and 4/5 animals, respectively, after 21 d. The acute reactions were no longer visible after 14 d. Under the test conditions, read across substance was considered to be irritating to rabbit skin (Kästner, 1986). Based on the results of the read across study,the test substance, C10 -12 and C18 -unsatd. DEA, is also considered as irritating to rabbit skin.

Eye irritation:

Study 1: An in vitro study was conducted to determine the eye irritation potential of the test substance, C10-12 and C18 -unsatd. DEA, using Reconstructed human Cornea-like Epithelium (RhCE) test method, according to OECD 492 Guideline, in compliance with GLP. Three tissues of the EpiOcularTM tissue model were treated with the test substance, positive or negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) prior to topical application of approximately 50 µL of the neat test substance. Sterile water was used as negative control and methyl acetate as positive control. After 30 minutes exposure on the surface of EpiOcularTM reconstructed ocular epithelium, followed by a 12 post-treatment immersion and 2 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The percentage viability for the test substance was determined to be 22.497%, which is well below the threshold indicating irritation potential. Therefore,the study authors concluded the test substance as irritant to the human eye under the study conditions(XCELLR8, 2017).However, based on the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage), hence, the classification for the test substance is considered to be inconclusive.

Study 2: An in vitro study was conducted to determine the eye damage potential of the test substance, C10-12 and C18-unsatd. DEA, according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. In this study, eye damage of the test substance was tested through topical application for 10 minutes, followed by an incubation period of 120 minutes. The test substance was applied as is (750 µL) directly on top of the freshly isolated bovine cornea sample. Negative and positive control items 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 negative control responses for opacity and permeability was less than the upper limits of the laboratory historical range, indicating that the negative control did not induce irritancy on the corneas. The mean IVIS of the positive control (Ethanol) was 29.7, which was marginally lower than the criteria range set for an acceptable test. As the score was only marginally lower, it was decided that the result was acceptable as the positive control group still provided its intended function, which was to show the sensitivity of the test system to a known ocular irritant. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The IVIS score of the test substance was determined to be 62.7 after 10 minutes of treatment, which is above the IVIS threshold of 55, indicating Category 1 conclusion. Under the study conditions, the test substance was concluded to cause serious eye damage (Envigo, 2018).

Justification for classification or non-classification

Skin irritation:

Based on the results of in vitro study with the test substance and a read across in vivo skin irritation study, the test substance, C10 -12 and C18 -unsatd. DEA, is considered to be irritant to skin with a classification as Skin Irritant 2; H315 - Causes skin irritation according to EU CLP criteria (Regulation 1272/2008/EC).

Eye irritation:

Based on the results of in vitro eye irritation studies, the test substance, C10 -12 and C18 -unsatd. DEA, is considered to be corrosive to eyes with a classification as Eye Damage 1; H318 - Causes serious eye damage according to EU CLP criteria (Regulation 1272/2008/EC).