Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Skin corrosion

reconstructed human epidermis (RhE) in vitro model EPISKIN™, OECD 431, GLP: negative

Skin irritation

reconstructed human epidermis (RhE) in vitro model EPISKIN™, OECD 439, GLP: negative

Eye irritation

reconstructed human cornea epidermis (RhCE) in vitro model EpiOcular™, OECD 492, GLP: negative

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:
2018-12-13 to 2018-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYÉI- National Institute of Pharmacy and Nutrition (21.04.2016)
Specific details on test material used for the study:
Storage 15-25 °C
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: 10-KERA-003 + 10-KERA-004 (normal human keratinocytes)
Source strain:
other: human
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
physiological saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small Model
- Tissue batch number(s): 18-EKIN-050

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (18-28°C)
- Temperature of post-treatment incubation (if applicable): 37±1°C in an incubator with 5±1% CO2 protected from light, ≥95% humidified atmosphere.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the incubation time the EpiSkin TMSM units were rinsed thoroughly with approximately 25 mL PBS 1x solution to remove the test item from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was dissolved to a final concentration of 3 mg/mL in saline buffer (1xPBS). The MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/mL.
- Incubation time: 3 hours (± 15 min)
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: at 570 nm (±10 nm; Read out range: 0-3.5 Abs)
- Linear OD range of spectrophotometer: Linearity range: 0.2136 – 3.1752

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean tissue viability is < 35 % after 3 min exposure (1A), the mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposure or the mean tissue viability is ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure (1B)
- The test substance is considered to be non-corrosive to skin if the mean tissue viability is ≥ 35 % after 4 hours exposure
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): An amount of 20 mg test item was applied evenly to the epidermal surface of the two test skin units/exposure times respectively. Subsequently, 100 μL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

NEGATIVE CONTROL / POSITIVE CONTROL
- Amount(s) applied (volume or weight): A volume of 50 μL positive control (glacial acetic acid) or negative control (NaCl 9 g/L) was applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 4 hours (±10 min) at room temperature (18-28°C).
Duration of post-treatment incubation (if applicable):
After the exposure of test item was terminated by rinsing with PBS, the EpiSkin units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere.
Number of replicates:
Two replicates were used for the test item and control(s) respectively.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
run 1 replicate 1
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
run 1 replicate 2
Value:
111
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Positive and negative controls showed the expected cell viability values within acceptable limits.
All assay acceptance criteria were met, the experiment was considered to be valid.

Table 1: OD values and cell viability percentages of the positive and negative control

Controls Optical Density (OD) Viability(%) Δ%
Negative Control:NaCl (9 g/L saline) 1 0.756 99 1.1
2 0.765 101  
mean 0.761 100
Positive Control: Glacial acetic acid 1 0.008 1 0.7
2 0.014 2  
mean 0.011 1

Table 2: OD values and viability percentages of the test item

Test Item Optical Density (OD) Viability(%) Δ%
Test substance 1 0.705 93 18.5
2 0.846 111  
mean 0.776 102
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The average test item treated tissue viability was 102 % at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure. The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.
Executive summary:

EpiSkinTM SM test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431 (29 July 2016) following GLP.

Disks of EPISKIN (two units / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1°C in an incubator with 5±1 % CO2 in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 102 % at 4 hours of exposure. Thus, the test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-04-03 to 2019-05-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum
Source strain:
other: human
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no-skin irritating test substances.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, (three-dimensional human epidermis model). One EpiSkin plate contains up to 12 reconstructed epidermis units (area: 0.38 cm2), ready-to-use
- Tissue batch number(s): 19-EKIN-015
- Expiry date: 15 April 2019
- Date of initiation of testing: 10 April 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: EpiSkin units are rinsed thoroughly with approximately 25 mL PBS 1x solutaion
- Observable damage in the tissue due to washing: Care is taken to avoid the damage of epidermis

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/L
- Incubation time: 3 h ± 5 min
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm (± 10nm; Read out range: 0-3.5 Abs)
- Linear OD range of spectrophotometer: 0.2136 – 3.1752

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test. Result: IC50 = 2.0 mg SDS/mL (range 1.5 - 3.0 mg/mL)
- Morphology: Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum. No. of cell layers: 8 (should exceed 4)
- Contamination: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure is greater than 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): First, 5 µL of deionised water will be applied to the epidermal surface in order to improve further contact between powder and epidermis. Subsequently, 10±2 mg of the test item will be applied evenly to the epidermal surface.

NEGATIVE CONTROL / POSITIVE CONTROL
- Concentration (if solution): A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) will be applied on the skin surface by using a suitable pipette. Chemicals may be spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42 hours (± 1h)
Number of replicates:
3 replicates for test item and controls
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1/ replicate 1
Value:
107
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1/ replicate 2
Value:
105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1/ replicate 3
Value:
114
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
109
Vehicle controls validity:
not applicable
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: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: was demonstrated in a seperate study

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD value between 0.6-1.5 and SD of the % viability ≤ 18
- Acceptance criteria met for positive control: yes, between 0-40 % of the negative control and SD of the % viability ≤ 18
- Acceptance criteria met for variability between replicate measurements: yes, SD of the % viability ≤ 18

Table: OD values and viability percentages of the controls and test item.

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

0.964

104

2

0.855

92

3

0.965

104

mean

0.928

100

standard deviation (SD)

6.85

Positive Control:
SDS (5 % aq.)

1

0.081

9

2

0.064

7

3

0.069

7

mean

0.072

8

standard deviation (SD)

0.92

Test Item:
Test substance

1

0.995

107

2

0.971

105

3

1.062

114

mean

1.009

109

standard deviation (SD)

5.06

Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (EU GHS: No Category).
Executive summary:

EpiSkinTMSM test of the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to OECD Guideline 439 under GLP compliance.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (± 1h) in an incubator with 5±1% CO2, ≥95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 109 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (EU GHS No Category).

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-05-28 to 2019-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYÉI- National Institute of Pharmacy and Nutrition (22.05.2019)
Species:
human
Strain:
other: Keratinocyte strain 4F1188
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: 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. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 µL of 0.3% (v/v) Triton X-100). A certificate of quality as provided by the supplier is available.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was applied in its original form (approximately 50 mg on top of 0.6 cm² tissue; approx. 83.3 mg/cm²), no formulation was required.
Duration of treatment / exposure:
6 h (± 15 min)
Duration of post- treatment incubation (in vitro):
18 h (± 15 min)
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used : The three-dimensional RhCE tissue construct is produced using primary human epidermal keratinocytes i.e., EpiOcular™ OCL-200. The EpiOcular™ OCL-200 RhCE tissues construct is similar to the in vivo corneal epithelium three-dimensional structure. In this assay, the test item is applied to the surface of the cornea epithelial construct for a fixed period, removed, and the tissue allowed to express the resulting damage. Liquids and solids are treated with different exposure and post-exposure incubations. Two construct tissues are used for each treatment and control group. Relative tissue viability is determined against the negative control-treated constructs by the reduction of the vital dye MTT (3-[4,5 -dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide). A concurrent positive control is used with each assay.
- RhCE tissue construct used, including batch number :
EpiOcular™ (OCL-200-EIT)
Supplier: MatTek In Vitro Life Science Laboratories Mlynske Nivy 73, Bratislava, Slovakia
Lot No.: 30608
Expiry date: 30 May 2019
- Doses of test chemical and control substances used :
test item: 50 mg on top of 0.6 cm² tissue (approx. 83.3 mg/cm²)
positive nd negative control: 50 µg on top of 0.6 cm² tissue
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
exposure: 6 h (± 15 min) (37±2°C in an incubator with 5±1% CO2, >95% humidified atmosphere)
post-exposure immersion incubation: 25 ± 2 min at room temperature
post-exposure incubation: 18 hours ± 15 minutes (37±2°C in an incubator with 5±1% CO2, >95% humidified atmosphere)
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570 nm ±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 - 3.1752
- Description of the method used to quantify MTT formazan : The precipitated formazan was quantified spectrophotometrically
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : according to guideline
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : historical OD negative control (range 1.138 - 2.464; mean 1.811); historical OD positive control (range 0.064 - 0.544; mean 0.261)
- Complete supporting information for the specific RhCE tissue construct used :
Biological contaminants: no biological contaminants were detected (HIV-1 virus, Hepatitis B virus, Hepatitis C virus, bacteria, yeast and other fungi)
Tissue viability (MTT QC assay, 1 h, n=3): OD (540-570 nm) = 1.296 ± 0.038
Barrier function (ET-50 assay, 100 µL 0.3 % Triton X-100, 3 time-points, n=2, MTT assay): ET-50 = 23.66 min
- Reference to historical data of the RhCE tissue construct
Tissue viability (MTT QC assay, 1 h, n=3): OD (540-570 nm) = 1.1 - 3.0 -> pass
Barrier function (ET-50 assay, 100 µL 0.3 % Triton X-100, 3 time-points, n=2, MTT assay): ET-50 = 12.2 - 37.5 min -> pass
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.
- Positive and negative control means and acceptance ranges based on historical data :
test positive control (mean OD: 0.233 - mean viability: 11 %), historical OD positive control (range 0.064 - 0.544; mean 0.261)
test negative control (mean OD: 2.050 - mean viability 100 %), historical OD negative control (range 1.138 - 2.464; mean 1.811)
- Acceptable variability between tissue replicates for positive and negative controls : < 20%
- Acceptable variability between tissue replicates for the test chemical: < 20%
Irritation parameter:
other: tissue viability
Run / experiment:
mean [% ]
Value:
90
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: tissue viability
Run / experiment:
replicate 1 [%]
Value:
92
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: tissue viability
Run / experiment:
replicate 2 [%]
Value:
87
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

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, acceptable OD 0.8 - 2.5 (test mean OD of negative control = 2.050)
- Acceptance criteria met for positive control: yes, acceptable percentage viability for positive control < 50 % (test mean viability of positive control: 11 %)
- Range of historical values if different from the ones specified in the test guideline:
historical OD positive control (range 0.064 - 0.544; mean 0.261) - historical viability positive control (range 3 - 41 %; mean 15 %)
historical OD negative control (range 1.138 - 2.464; mean 1.811)
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is, thus, considered as non-irritant to eye (UN GHS No Category).
Executive summary:

The acute ocular irritation potential of the test item was determined in the three-dimensional RhCE tissue in the EpiOcular™ model in vitro according to OECD Guideline 492 following GLP.

Before treatment the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2°C in an incubator with 5 ± 1% CO2, >95% humidified atmosphere). Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37 ± 2°C in an incubator with 5 ± 1% CO2, >95% humidified atmosphere and protected from light. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 90 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to eye. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is, thus, considered as non-irritant to eye (UN GHS No Category).

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

OECD 431

EpiSkinTM SM test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431 (29 July 2016) following GLP.

Disks of EPISKIN (two units / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1°C in an incubator with 5±1 % CO2 in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 102 % at 4 hours of exposure. Thus, the test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.

Skin irritation - in vitro

OECD 439

EpiSkinTMSM test of the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to OECD Guideline 439 under GLP compliance.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1°C for 42 hours (± 1h) in an incubator with 5±1% CO2, ≥ 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5% aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 109 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Eye irritation - in vitro

OECD 492

The acute ocular irritation potential of the test item was determined in the three-dimensional RhCE tissue in the EpiOcular™ model in vitro according to OECD Guideline 492 following GLP.

Before treatment the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2°C in an incubator with 5 ± 1% CO2, >95% humidified atmosphere). Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37 ± 2°C in an incubator with 5 ± 1% CO2, >95% humidified atmosphere and protected from light. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 90 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to eye. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is, thus, considered as non-irritant to eye (UN GHS No Category).

Justification for classification or non-classification

Based on the result of the OECD in vitro skin and eye irritation studies following GLP, the registered substance is not classified in accordance with Regulation (EC) No 1272/2008.