Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/corrosion:

OECD 439 (RL1): The test item is not irritating to skin. 

OECD 431 (RL1): The test item is not corrosive to skin. 

Study similar to OECD 404 (RL4): The study cannot be used for classification and labelling according to Regulation (EC) No 1272/2008.

Eye irritation/corrosion

OECD 492 (RL1): No UN GHS category  

Study similar to OECD 405 (RL4): The study cannot be used for classification and labelling according to Regulation (EC) No 1272/2008.

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:
experimental study
Adequacy of study:
supporting study
Study period:
22 - 25 Feb 1982
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Abraded skin, occlusive, no individual scores treatment duration, animal number
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted in 2015
Deviations:
yes
Remarks:
Abraded skin, occlusive, no individual scores treatment duration, animal number
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: in-house breeding (Hoechst Aktiengesellschaft, Pharma Forschung, Toxikologie, Germany)
- Weight at study initiation: 1.73 - 2.43 kg
- Housing: individual
- Diet: ERKA 8300 (Futtermittelwerk Robert Koch oHG, Hamm/Westf., Germany), ad libitum
- Water: ad libitum
Type of coverage:
occlusive
Preparation of test site:
other: half intact / half abraded
Vehicle:
unchanged (no vehicle)
Remarks:
wetted with 0.75 mL polyethyleneglycol 400
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
Duration of treatment / exposure:
24 h
Observation period:
72 h after patch removal
Number of animals:
6
Details on study design:
TEST SITE
- Area of exposure: 2.5 x 2.5 cm²
- Type of wrap if used: The test item was applied onto a surgical gauze pad (Hansamed Wundpflaster). The test item and gauze pad were then covered with an impermeable polyethylene foil (width: 6 - 8 cm). The entire trunk of each animal was then encased in an elastic wrapping (polyurethan-warp thread bandage (Dauerbinde K)).

OBSERVATION TIME POINTS
0, 24 and 48 h after patch removal

SCORING SYSTEM: according to §1500.41 of the Federal Register 38, No. 187, 27.9.1973, p 27019 (comparable to Draize scoring system)
- Method of calculation: The skin irritation index was calculated per animal. The erythema and edema scores after 0 and 48 h after patch removal were added for the intact and abraded skin. Finally the sum of the scores of all animals was divided through the amount of animals and through 4.
Irritation parameter:
other: skin irritation index
Basis:
mean
Remarks:
of 6 animals
Time point:
other: 0 h / 48 h after patch removal
Score:
0.1
Max. score:
8
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No further data was given in the study report.
Interpretation of results:
study cannot be used for classification
Conclusions:
CLP: based on the available data, no classification is possible according to Regulation (EC) No 1272/2008. A conclusion regarding the classification or non-classification can only be made in combination with additional data.
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 - 22 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
No information provided on technical proficiency; no positive control data after 3 min exposure
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
No information provided on technical proficiency; no positive control data after 3 min exposure
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 Bis (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted in 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container closed and dry in a cool, well-ventilated place, protected from light
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human derived non-transformed epidermal keratinocytes
Source strain:
other: not applicable
Justification for test system used:
The reconstructed human epidermis model test method is an accepted in vitro method to replace in vivo animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.
Vehicle:
unchanged (no vehicle)
Remarks:
; the epidermis surface was wettened with 20 ± 2 µL of deionised water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Skin Ethic™ RHE-model RHE/S/17, manufactured by EPISKIN/Skin Ethic Laboratories, Lyon, France
- Tissue batch number: 17-RHE-021
- Date of initiation of testing: 21 Feb 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume of washing steps: gently rinsed with approx. 20 mL DPBS solution


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h (± 15 min)
- Spectrophotometer: 96-well plate microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 570 = 1.3 (OD 570 historic negative control: 1.881 (3 min exposure) and 2.001 (1 h exposure)
- Barrier function: 4.6 h
- Morphology: 5.5 cell layers, well-differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean tissue viability after 3 min exposure is < 50%, or the mean tissue viability is ≥ 50% after 3 min exposure and < 15% after 1 h exposure.
(If the mean tissue viability after 3 min exposure is < 18% the substance is considered as corrosive Sub-categories 1A and if it is ≥ 18% the substance is considered as combination of optional Sub-categories 1B and 1C).
- The test substance is considered to be non-corrosive to skin if the viability is ≥ 50% after 3 min exposure and ≥ 15% after 1 h exposure.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 20 ± 3 mg

NEGATIVE CONTROL
- Amount(s) applied: 40 ± 3 µL
- Concentration: neat/ undiluted

POSITIVE CONTROL
- Amount(s) applied: 40 ± 3 µL
- Concentration: 8N
Duration of treatment / exposure:
3 min and 1 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of test item (100%), 3 min exposure
Value:
100.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value of test item (100%),1 h exposure
Value:
108.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: no information given

ACCEPTANCE OF RESULTS:
The mean OD 570 value of the negative control tissues should be between 0.8 and 3. The negative controls should be higher or equal to a historical established boundary (here: two standard deviations below the current historical mean = 1.635 (1.881 - 2 x 0.123) and 1.429 (2.001 - 2 x 0.286) for 3 min and 1 h exposure respectively.

The positive controls should be lower or equal to a historical established boundary (here: three standard deviations above the current historical mean = 0.99% (0.58 + 3 x 0.14).

The range between identically treated tissues viability should be < 30% with the exception of cases with ODs ≤ 0.3 and for viabilities out the range 20 - 100%.

- Acceptance criteria met for negative control: Yes. The mean OD value of the two negative control tissues was in the range of 0.8 - 3 at each exposure time (2.238 and 2.020 at 3 min and 1 h exposure, respectively).
- Acceptance criteria met for positive control: Yes. The positive control treatment resulted in a viability of < 0,99% (0.6% at 1 h exposure).
- Acceptance criteria met for variability between replicate measurements: Yes. In the range 20 - 100 % viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates did not exceed 30% in any case (negative control: 9% after 3 min exposure and 0.6% after 1 h exposure, treatment group: 10.5% and 2.1% after 3 min and 1 h exposure, respectively). For the positive control the threshold of 30% was exceeded (40%), but as the OD 570 was < 0.3 it is not considered to be a deviation.

Table 1: MTT assay after 3 min exposure

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD570

2.142

2.334

-

-

2.138

2.361

OD570 (mean values of replicates)

2.238

-

2.25

Viability (%)

100

-

100.5

Table 2: MTT assay after 1 h exposure

Negative control

Positive control

Test item

Tissue sample

1

2

1

2

1

2

OD570

2.027

2.013

0.01

0.014

2.174

2.22

OD570 (mean values of replicates)

2.02

0.012

2.197

Viability (%)

100

0.60

108.8

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model according to OECD Guideline 431. 

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues.

Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 +/- 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide) were applied to the tissues. Before application of 20 +/- 3 mg of the solid test item, 20 +/- 2 µL of deionised water was spread to the epidermis surface to improve the contact between test item and the epidermis. 

After treatment with the positive control, the mean viability value was 0.6% and, thus, lower than the historically established threshold of 0.99%. After treatment with the negative control (deionised water) the mean ODs were 2.238 (3 minute exposure) and 2.020 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.635 and 1.429, respectively. Thus, the acceptance criteria were met. 

Following treatment with the test item, the tissue viability was >= 50% after 3 minutes exposure (mean viability: 100.5%) and >= 15% after 1 hour exposure (mean viability: 108.8%), i.e. according to OECD 431 the test item is not considered as corrosive to skin. 

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 06 - Apr 16, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
June 18, 2019 (corrected June 26, 2020)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment; the test item was applied undiluted.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
standard model
Vehicle:
unchanged (no vehicle)
Remarks:
No vehicle used in this study; The test item was applied neat to the tissues.
Details on test system:
CELL CULTURE
- Supplier: Episkin/SkinEthic Laboratories, Lyon, France
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 21-RHE-031


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL PBS using a pipette. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)
Duration of treatment / exposure:
42 min (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Experiment 1 / Run 1
Value:
102
Vehicle controls validity:
not applicable
Remarks:
The test item was applied neat to the tissues
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

- Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The OD values for the negative control shall be in the range of ≥ 0.8 and ≤ 3.0 as given in OECD Guideline 439. The values obtained were: 1.755 to 1.852

- Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:
The negative control data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 1.2 at 570 nm. The standard deviation value is considered valid if ≤ 18% of group mean-value.
The reults obtained were: Mean OD = 1.816, SD =1.5%, group mean SD values: 6.7% (positive control) 3.0% (negative control)


The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is < 40%. The standard deviation value is considered valid if ≤ 18% of group mean-value.







 

 Group Time / [min]  Mean OD  Mean Relative viability / [%]
 Negative Control 42  1.816 100 
 Positive Control 42

0.027

1.5

 Test Material

42

1.852

102

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item Diazo PW 980 is not considered to possess an irritant potential to skin (UN GHS: No Category).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Moistened substance, scoring system, animal number, no individual scores given
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
adopted in 2012
Deviations:
yes
Remarks:
Moistened substance, scoring system, animal number, no individual scores given
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: in-house breeding (Hoechst Aktiengesellschaft, Pharma Forschung, Toxikologie, Germany)
- Weight at study initiation: 2.2 - 4.0 kg
- Housing: individual
- Diet: ERKA 8300 (Futtermittelwerk Robert Koch oHG, Hamm/Westf., Germany), ad libitum
- Water: ad libitum

Vehicle:
unchanged (no vehicle)
Remarks:
wetted with 0.15 mL polyethyleneglycol 400
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
24 h
Reading time points: 1, 7, 24, 48 and 72 h
Observation period (in vivo):
72 h
Number of animals or in vitro replicates:
6
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): with physiological saline
- Time after start of exposure: 24 h

SCORING SYSTEM: classification according to "Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics" FDA, Austin, Texas, p 51, 1975 (for further details refer to section "any other information on materials and methods incl. tables")
- Method of calculation: The eye irritation index was calculated per animal by summing up the different scores for cornea opacity, iris and conjuctivae 1, 7, 24, 48 and 72 h after application. Finally the mean index of all animals was calculated and the highest index (out of the different time points) was chosen for classification.

TOOL USED TO ASSESS SCORE: magnifier / fluorescein sodium (0.01%)
Irritation parameter:
other: eye irritation index
Basis:
mean
Remarks:
of 6 animals
Time point:
other: 1 h
Score:
11
Max. score:
110
Reversibility:
not specified
Irritant / corrosive response data:
The test substance was slightly irritant according to the applied scoring system. No further data was given in the study report.
Interpretation of results:
study cannot be used for classification
Conclusions:
CLP: based on the available data, no classification is possible according to Regulation (EC) No 1272/2008. A conclusion regarding the classification or non-classification can only be made in combination with additional data.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 - 06 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted in 2015
Deviations:
yes
Remarks:
No information on historical data given.
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace in vivo animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcularTM human cell construct (OCL-200, OCL-212, MatTek Corporation) is used in this assay. It is a nonkeratinized epithelium prepared from normal human keratinocytes. All biological components of the EpiOcularTM tissue and the kit culture medium have been tested and are free of contamination. An analysis for tissue functionality and quality was performed: the barrier function, cell viability were meeting the acceptance criteria as well as the histological appearance of the cells.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 mg
- Concentration: undiluted
Duration of treatment / exposure:
6 h ± 15 min
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
25 ± 2 min (Post-exposure immersion incubation)
18 h ± 15 min (Post-treatment incubation)
Number of animals or in vitro replicates:
2 replicates
Details on study design:
- Details of the test procedure used
The cytotoxicity of the test item (and thus the ocular irritation potential) is evaluated by measuring the relative viability of the treated RhCE tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT in control and test item-treated cultures.

- RhCE tissue construct used, including batch number
The EpiOcularTM human cell construct (OCL-200 and OCL-212, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 23769)

- Doses of test chemical and control substances used
50 mg test item; 50 μL positive control (methyl acetate) and 50 μL negative control (sterile deionized water)

- Wavelength used for quantifying MTT formazan
570 nm

- Description of the method used to quantify MTT formazan
Inserts were removed from the 24-well plate after the incubation time in MTT solution (3 h ± 10 min), the bottom of the insert was blotted on absorbent material and transferred to a 6-well plate containing 2 mL isopropanol per well in a manner avoiding the isopropanol to flow into the insert. The plate was sealed with standard plate sealer. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2-3 h at room temperature. 200 μL samples from each tube were placed into the wells of a 96-well plate and the absorbance/optical density was measured in a 96-well plate spectrophotometer to determine cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test item requires classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60% of the negative control.
The test item requires no classification and labelling according to UN GHS (No Category), if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
No information is given on historical data, but the results obtained for the negative and positive control meet the acceptance criteria given in the OECD guideline 492.

- Complete supporting information for the specific RhCE tissue construct used
The barrier function integrity test resulted in an ET50 of 4.6, cell viability was determined to have an OD of 1.3, the RHE model was tested to be a well differentiated epidermis with 5.5 cell layers without any significant histological abnormality, thus meeting the acceptance criteria.

- Reference to historical data of the RhCE tissue construct
no further information available in the study report

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
no information provided in the study report

- Positive and negative control means and acceptance ranges based on TG OECD 492
negative control mean OD: 1.381 (acceptance range: 0.8 - 2.5)
positive control mean viablity: 11.2% (acceptance criteria: < 50% of the negative control)

- Acceptable variability between tissue replicates for positive and negative controls
The variability between tissue replicates for positive and negative controls meets the acceptance criteria of < 20%.

- Acceptable variability between tissue replicates for the test chemical
The variability between tissue replicates for the test chemical meets the acceptance criteria of < 20%.
Irritation parameter:
other: mean viability (%)
Run / experiment:
negative control
Value:
100
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: mean viability (%)
Run / experiment:
positive control
Value:
11.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: mean viability (%)
Run / experiment:
test item
Value:
67.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.381) of the two negative control tissues lies between 0.8 and 2.5.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (11.2%) lies below 50% of the control viability.
- The variability between tissue replicates for positive, negative controls and test chemical were 1.9%, 17.4% and 12.1% respectively, thus meeting the acceptance criteria of < 20%.

The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer. In the pre-test medium coloration by the test item was observed, but no tissues were stained during the study. Therefore, no additional tissues for color control were treated.

Table 1: MTT results of eye irritation study

Controls

 

Optical Density (OD)

Viability (%)

Δ%

Negative Control: Sterile deionized water

1

1.261

91.3

17.4

2

1.501

108.7

mean

1.381

100.0

-

standard deviation (SD)

12.30

 

Positive Control:
Methyl acetate

1

0.141

10.2

1.9

2

0.167

12.1

mean

0.154

11.2

-

standard deviation (SD)

1.34

 

Test item

1

1.015

73.5

12.1

2

0.848

61.4

mean

0.932

67.5

-

standard deviation (SD)

8.56

-

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye hazard potential.
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. 

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. 

Duplicates of the EpiOcular-model were treated with the test item, the negative or the positive control for 6 hours (+/- 15 min). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. 

After treatment with the negative control (sterile deionized water) the mean OD was 1.381 (study acceptance criterion: > 0.8 and < 2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 11.2% (study acceptance criterion: < 50%). Thus, the acceptance criterion were met. 

Following treatment with the test item, the tissue viability was 67.5% and, thus, higher than 60%, i.e. according to OECD 492 the test item did not show an eye hazard potential. 

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the data, the test item is not classified for skin/eye irritation according to Regulation (EC) No 1272/2008.