Registration Dossier

Administrative data

Description of key information

Skin corrosion in vitro: The test item was considered to be non-corrosive to skin because cell viability was determined to be 97.9 % after 3 minutes and 96.5 % after 60 minutes (OECD 431; EpiDerm Human Skin Model).

 

Skin irritation in vitro: The test item was considered to be non-irritant to skin because the relative mean viability of the test item treated tissues was determined to be 95.4 % after the 15-minute exposure period and 42-hours post-exposure incubation (OECD 439; EPISKIN reconstructed human epidermis model).

 

Eye damage/irritation in vitro: The test item was found to be non-hazardous to the eye because the in vitro irritancy score (IVIS) was 0.3 in the BCOP assay (OECD 437 and EU Method B.47) and corrected viability of tissues was 114.1 % using the Human Cornea Model test (OECD 492).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September 2016 to 30 September 2016
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
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: neonatal
Vehicle:
unchanged (no vehicle)
Details on test system:
PURPOSE OF THE TEST
- The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes.
- Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
- This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 μL
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Duplicate tissues
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
97.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean relative viability (% of negative control)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60-minute exposure
Value:
96.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean relative viability (% of negative control)
Other effects / acceptance of results:
DIRECT MTT REDUCTION
- The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item did not become coloured.
- This was taken to indicate the test item did not have the potential to cause colour interference.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- Mean OD562 values and viabilities for the negative control, positive control and test item are given in Appendix 1 (attached).
- The relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- The mean OD562 for the negative control treated tissues was 1.679 for the 3-Minute exposure period and 1.850 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 4.7 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

RELATIVE MEAN VIABILITIES FOR EACH TREATMENT GROUP

Exposure period

Percentage viability negative control*

Percentage viability positive control

Percentage viability test item

3 minute

100

5.9

97.9

60 minutes

100

4.7

96.5

* Mean viability of the negative control tissues is set at 100%

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean viability of the test item treated tissues was 97.9 % after 3 minutes exposure and 96.5 % after 60 minutes exposure.
Executive summary:

GUIDELINE

The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and cytotoxicity is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

METHODS

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The

optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 5.9 % (positive control and 97.9 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 4.7 % (positive control) and 96.5 % (test item).

 

CONCLUSION

The relative mean viability of the test item treated tissues was 97.9 % after 3 minutes exposure and 96.5 % after 60 minutes exposure.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2016 to 24 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
increase in post-exposure incubation time with no impact on integrity or validity of the test (see below)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
increase in post-exposure incubation time with no impact on integrity or validity of the test (see below)
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Source strain:
other: adult
Vehicle:
unchanged (no vehicle)
Details on test system:
PURPOSE OF THE TEST
- The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 1 hours (Fentem et al., 2001, Zuang et al., 2002, Cotovio et al., 2005, Portes et al., 2002 and Hartung, 2007). The actual post-exposure incubation period was 43.75 hours and was recorded as a deviation in the study file. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-hour post-exposure incubation period may also be determined for
test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.
- The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Following a full validation study, the EpiSkin reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between irritating and non-Irritating test items.
- The procedure followed was based on the recommended EpiSkin SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.
- Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
43.75 hours.(see deviations from study plan, below)
Number of replicates:
Triplicate tissues
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test
Value:
95.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: relative mean viability of tissues after 15-minute exposure and 43.75-hour post-exposure incubation
Other effects / acceptance of results:
DIRECT MTT REDUCTION
- The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item was colourless.
- It was therefore unnecessary to run colour correction tissues.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Appendix 1 (attached). The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Appendix 1.
- The relative mean viability of the test item treated tissues was 95.4 % after a 15-minute exposure period and 43.75-hour post-exposure incubation period.
- It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

QUALITY CRITERIA
- The relative mean tissue viability for the positive control treated tissues was 11.6 % relative to the negative control treated tissues and the standard deviation value of the viability was 0.5 %. The positive control acceptance criteria were therefore satisfied.
- The mean OD562 for the negative control treated tissues was 1.115 and the standard deviation value of the viability was 6.8 %. The negative control acceptance criteria were therefore satisfied.
- The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 4.5 %. The test item acceptance criterion was therefore satisfied.
Interpretation of results:
GHS criteria not met
Conclusions:
Relative to the concurrently treated negative control the viability of the test item treated tissues was 95.4 %.
Executive summary:

GUIDELINE

The study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (adopted 28 July 2015) and Method B.46 in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of the test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 ± 1 hours.The actual post-exposure incubation period was 43.75 hours and was recorded as a deviation in the study file.The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

 

METHODS

Triplicate tissues were treated with discs of the test item for an exposure period of 15 minutes. At the end of the exposure period, each tissue was rinsed before incubating for 43.75 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data were presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

The relative mean viability of the test item treated tissues was 95.4 % after the 15-minute exposure period and 43.75 hour post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

CONCLUSION

Relative to the concurrently treated negative control the viability of the test item treated tissues was 95.4 %.

Endpoint:
skin irritation: in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 2016
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
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).
- Excised eyes were transported to the test facility over ice packs on the same day of slaughter.
- 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):
120 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
REFERENCE ITEM PREPARATION
- The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.
- The positive control item (ethanol) was used as supplied.

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 °C 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 (see Annex 1, attached). The average opacity for all corneas was calculated.
- Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

TREATMENT OF CORNEAS
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the cornea 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 °C for 10 minutes.
- At the end of the exposure period the test item 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 °C for 90 minutes.

PERMEABILITY DETERMINATIONS
- After incubation the medium in the posterior chamber of each holder was decanted and retained.
- Medium representing each cornea (360 μL) was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

HISTOPATHOLOGY
- The corneas were retained after testing for possible conduct of histopathology.
- Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface.
- The cassette was immersed in 10% neutral buffered formalin.

DATA EVALUATION
- Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

OPACITY MEASUREMENT
- The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading.
- These values were then corrected by subtracting the average change in opacity observed for the negative control
corneas.
- The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT
- The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea.
- The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

VISUAL OBSERVATION
- The condition of the cornea was visually assessed post treatment.

CRITERIA FOR AN ACCEPTABLE TEST
- Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 52.0.
- Sodium chloride solution (0.9% w/v) was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤ 2.9 and for permeability ≤ 0.103.

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.
Irritation parameter:
in vitro irritation score
Run / experiment:
Main test
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritant / corrosive response data:
CORNEAL OPACITY AND PERMEABILITY MEASUREMENTS
- Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1 (attached).

CORNEAL EPITHELIUM CONDITION
- The condition of each cornea is given in Appendix 2 (attached)
- The corneas treated with the test item were clear post-treatment and post-incubation.
- The corneas treated with the negative control item were clear post-treatment and post-incubation.
- The corneas treated with the positive control item were cloudy post-treatment and post-incubation.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Scores were determined to be 0.3 for the test item, 0.9 for the negative control and 43.7 for the positive control.

CRITERIA FOR AN ACCEPTABLE TEST
- The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.
- The negative control gave opacity of ≤ 2.9 and permeability ≤ 0.103. The negative control acceptance criteria were therefore satisfied.
Interpretation of results:
GHS criteria not met
Conclusions:
In Vitro Irritancy Scores were reported as 0.3 for the test item, 0.9 for the negative control and 43.7 for the positive control.
Executive summary:

GUIDELINE

The study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay”and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

METHODS

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. 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).

 

RESULTS

In Vitro Irritancy Scores were reported as 0.3 for the test item, 0.9 for the negative control and 43.7 for the positive control.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 January 2017 to 20 January 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:
human
Details on test animals or tissues and environmental conditions:
PURPOSE OF STUDY
- Eye irritation is generally defined as "the production of reversible changes in the eye". The potential for chemical induced eye irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. It is usually determined in vivo in the Draize rabbit eye irritation test as described in OECD guideline 405. In a pre-validation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the EpiOcular human cornea model and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.
- The EpiOcular Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
- The test consists of a topical exposure of a human reconstructed cornea model to the neat test item followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 μL
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Duplicate tissues
Details on study design:
TEST ITEM PREPARATION
- Test item (50 µL) was dispensed directly onto duplicate EpiOcular tissue for 30 minutes.

EPIOCULAR KIT COMPONENTS
- Lot number: 23759
- Sealed 24-well plate: Contains 12/24 inserts with EpiOcular tissues on agarose
- Serum-free test medium: DMEM-Medium
- Positive control: Methyl Acetate (CAS#79-20-9)
- 12-well plate: Holding plate
- 24-well plates: For MIT viability assay
- 6-well plates: For storing inserts, or for topically applying test agents
- Ca++Mg++-Free D-PBS: Dulbecco's Phosphate Buffered Saline

MTT-100 ASSAY KIT COMPONENTS
- 1 vial (2 mL): MIT concentrate
- 1 vial (8 mL): MIT diluent (supplemented DMEM) for diluting MIT concentrate prior to use in the MTT assay
- 1 bottle (60 mL): Extractant solution (lsopropanol) for extraction of formazan crystals

CELL CULTURE
- EpiOcular kits and MTT-100 kits were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organised basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular tissues (surface 0.6 cm2) were cultured on specially prepared cell culture inserts (MILLICELL ,10 mm Ø).
- EpiOcular tissues were shipped on 17 January 2017 at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt of the EpiOcular tissues, they were stored until next day in the refrigerator at 2 to 8 °C. Next day the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
- Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70 % isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the assay medium. After one hour, the assay medium was replaced by 1 mL of fresh assay medium at 37 °C and the EpiOcular tissues was incubated at standard culture conditions overnight (about 17 hours).

MTT SOLUTION
- On the day of the experiment, an MTT solution of 1 mg/mL in DMEM was prepared.

ASSESSMENT OF DIRECT MTT REDUCTION BY THE TEST ITEM
- Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose, approximately 50 μL of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
- Since the MTT solution colour did not turn blue/purple, the test item is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.

ASSESSMENT OF COLOURED OR STAINING MATERIALS
- Coloured test items or test items which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colorant bound to the tissue and would be extracted together with MTT. Therefore, each test item had to be checked for its colorant properties.
- Since the test item was non-coloured additional tests had to be performed to assess, if it becomes colourant after contact with water or isopropanol. For this purpose, each 50 μL of the test item were added to 1.0 mL of water and to 2 mL isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least one hour, the isopropanol mixture for 3 hours at room temperature.
- Since the test item did not dye water or isopropanol, additional tests with viable tissues did not have to be performed.

EXPERIMENTAL PROCEDURE
- Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 µL topically on the EpiOcular tissues. The tissues were incubated at standard culture conditions for 30 minutes.
- At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilised a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium (room temperature) in a pre-labelled 12-well plate for 12-minute immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue.
- At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 mL of warm assay medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT ASSAY
- After post-treatment incubation of 120 minutes the MTT assay was performed.
- At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
- Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight (about 17 hours) at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
- The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
- The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

DATA RECORDING
- The data generated were recorded in the laboratory protocol. The results were presented in tabular form, including experimental groups with the test item and the controls.

DATA EVAUATION
- The mean OD value of the blank control wells (OD Blk) for each experiment was calculated.
- The mean OD Blk was subtracted from each mean OD value of the same experiment (blank corrected values).
- The mean value of the two aliquots for each tissue (= corrected test item OD) was calculated.
- The mean value of the two relating tissues for each control and test item (= corrected mean OD) was calculated. For further calculations only the corrected mean negative control OD value was needed.
- The corrected OD value of the negative control corresponds to 100% viability (corrected negative control OD = Negative Control OD – OD Blk = 100% Viability).

CALCULATIONS FOR VIABILITY TESTS ONLY
- The percentage viability of each of the two relating tissues was calculated relative to the negative control (100 % control) for each control and test item using the equation viability [%] = 100 x corrected test item OD / corrected mean negative control OD.
- The difference in viability between duplicate tissues was calculated. The test is considered invalid if the difference is > 20 %.
- The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model.

PREDICTION MODEL
- If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant.
- If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.
- A single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60 ± 5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.
- Since the results of the test were unequivocal, a second test was not requested.

ACCEPTABILITY OF ASSAY
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5.
2) The mean relative viability of the positive control is below 50 % of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: cell viability
Remarks:
percent
Run / experiment:
Main test
Value:
114
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: threshold for irritancy: ≤ 60%)
Other effects / acceptance of results:
RESULTS
- Results after treatment for 30 minutes with test item and the controls are shown in the attached table.
- The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water and isopropanol did not lead to a change in colour.
- Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.
- The mean relative absorbance value of the test item, corresponding to the cell viability, did not decrease (114.1%; threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye.

ACCEPTANCE CRITERIA
- The negative control OD was > 0.8 and < 2.5 (values between 1.597 and 1.827).
- The mean relative viability of the positive control was below 50 % of the negative control viability (12.9 %).
- The difference of viability between the two relating tissues of a single item was < 20% (values between 2.0 % and 11.8 %) in the same run (for positive and negative control tissues and tissues of single test items).
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions reported, the test item does not possess any eye irritating potential.
Executive summary:

GUIDELINE

An in vitro study was conducted to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The investigation was performed in compliance with the OECD Guideline for Testing of Chemicals 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July 2015) and the MatTek Corporation Protocol: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular Model (29 June 2015).

METHODS

The colourless test item did not prove to be an MTT reducer in the MTT pre-test, and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed. Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes.

 

RESULTS

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). Irritating effects were not observed following incubation with the test material. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease (114.1%).

 

CONCLUSION

Under the experimental conditions reported, the test item does not possess any eye irritating potential.

Endpoint:
eye irritation: in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin corrosion in vitro

The key study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B.40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and cytotoxicity is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 5.9 % (positive control and 97.9 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 4.7 % (positive control) and 96.5 % (test item).

 

Skin irritation in vitro

The key study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (adopted 28 July 2015) and Method B.46 in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of the test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42±1 hours. The actual post-exposure incubation period was 43.75 hours and was recorded as a deviation in the study file. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

 

Triplicate tissues were treated with discs of the test item for an exposure period of 15 minutes. At the end of the exposure period, each tissue was rinsed before incubating for 43.75 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data were presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was 95.4 % after the 15-minute exposure period and 43.75 hour post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

Skin irritation in vivo

Skin irritation does not need to be investigated in vertebrate animals because the test material was demonstrated to be non-hazardous using validated in vitro methods (cell viability ≥ 50 % after 3 minutes and ≥ 15 % after 60 minutes using OECD 431 and percentage tissue viability after exposure and post-treatment incubation > 50 % using OECD 439).

 

Eye irritation in vitro – BCOP assay

The study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. 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 In Vitro Irritancy Scores were reported as 0.3 for the test item, 0.9 for the negative control and 43.7 for the positive control.

Eye irritation in vitro Human cornea model test

An in vitro study was conducted to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The investigation was performed in compliance with the OECD Guideline for Testing of Chemicals 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July 2015) and the MatTek Corporation Protocol: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular Model (29 June 2015).

 

The colourless test item did not prove to be an MTT reducer in the MTT pre-test, and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed. Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes.

 

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). Irritating effects were not observed following incubation with the test material. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease (114.1%). Under the experimental conditions reported, the test item does not possess any eye irritating potential.

 

Eye irritation in vivo

Eye irritation potential does not need to be investigated in vertebrate animals because the test material was demonstrated to be non-hazardous (In vitro irritancy score ≤ 3) using the Bovine Corneal Opacity and Permeability (BCOP) assay and the Human Cornea Model test where viability of tissues was shown to be > 60 %).

Justification for classification or non-classification

Skin corrosion:The test item was considered to be non-corrosive to skin because cell viability was determined by OECD 431 (28 July 2015) to be > 50 % after 3 minutes and > 15 % after 60 minutes using the EpiDerm Human Skin Model. Classification for skin corrosivity in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

 

Skin irritation: The test item was determined by OECD 439 (28 July 2015) to be non-irritant to skin because percentage tissue viability after exposure and post-treatment incubation was found to be > 50 % using the EPISKIN reconstructed human epidermis model. Classification for skin irritation in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

Eye damage/irritation:The in vitro irritancy score (IVIS) was determined to be ≤ 3 for the test item in the BCOP assay and, in accordance with OECD 437 (26 July 2013), no classification is required under the terms of GHS, which is applied in the EU by Regulation (EC) No. 1272/2008 and subsequent amendments. The result was confirmed by the Human Cornea Model test in which viability of tissues was determined to be > 60 % relative to the negative control.