Registration Dossier

Diss Factsheets

Administrative data

Description of key information

OECD 439 (in vitro skin irritation test): positive results


OECD 431 (in vitro skin corrosion test): negative results


OECD 492 (in vitro eye irritation test): negative results


OECD 438 (ex vivo eye irritation test): negative results

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Aug 2019 to 10 Mar 2020
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:
06 July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
18 June 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified: origin: adult donors
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model / Human Epidermis SM/13
- Tissue batch number(s): Lot no.: 19-EKIN-033
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 13-08-2019 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: 14-08-2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37°C, 5% CO2; for 42 hours. For further information see below.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: not applicable

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3hrs at 37°C, 5% CO2
- Spectrophotometer: Labtech LT-4500
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: 10 nm
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD values (mean of triplicate tissues=0.705) for negative control are within the TG acceptability ranges of 0.6 - 1.5.
- Barrier function: IC50 = 2.1 mg/mL (within the acceptability range of 1.5-3.0 mg/mL
- Morphology: quality control performed by the supplier showed a Multi-layered, highly differentiated epidermis consisting of organised basal, spinous and granular layers, and a multilayered stratum corneum, 8.5 cell layers
- Reproducibility: historical data are provided in the report and demonstrated reproducibility over time (SD positive historical control = 0.071; SD positive historical control = 0.092)


NUMBER OF REPLICATE TISSUES: 3 (triplicate)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): water killed then the tissues were frozen for 6 months
- N. of replicates : 3
- Method of calculation used: This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues. As the results of the assay were positive without using the results of direct MTT reduction to perform quantitative correction, it was considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

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 irritating or corrosive to skin if the viability after 15-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if the viability after 15-minutes exposure / 42-hours incubation is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL: Dulbecco's Phosphate buffered saline with Ca2+ and Mg2+
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): used as supplied

POSITIVE CONTROL: Sodium dodecyl sulphate (SDS)
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% w/v aqueous solution (prepared within 2hrs of being applied to the test system)
- other: To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item).
Duration of treatment / exposure:
15 minutes at room temperature, after which each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++ to remove residual test item.
Duration of post-treatment incubation (if applicable):
Subsequently the skin tissues were incubated for 42 hours at 37°C, 5% CO2
Number of replicates:
Triplicate; treatment and concurrent negative control and positive control groups
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean (n=3)
Value:
32.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: It was considered unnecessary to perform IL-1α analysis. On the basis that the IL-1α analysis would not generate additional relevant information and there was an expectation of comparable results to the MTT relative mean viability endpoint results
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: The MTT solution containing the test item turned blue. An assessment found the test item was able to directly reduce MTT and an additional procedure using non-viable, water-killed, tissues was performed. However, as the results of the assay were positive without using the results of direct MTT reduction to perform quantitative correction, it was considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes..
- Colour interference with MTT: The solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD570 for the negative control treated tissues was 0.705 and the standard deviation value of the viability was 10.0%.
- Acceptance criteria met for positive control: Yes. The relative mean tissue viability for the positive control treated tissues was 3.8% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%.
- Acceptance criteria met for variability between replicate measurements: Yes. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 9.4%.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data. Historic Control Data (n=30): the mean OD of the positive control was 0.085 ; range 0.030 to 0.264. In this same period the mean OD of the negative control was 0.831 ; range 0.648 – 1.067.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item


 
























































Item



OD570 of tissues



Mean OD570 of triplicate tissues



± SD of OD570



Relative mean viability (%)



± SD of Relative mean viability (%)



Negative Control Item



0.680



0.705



0.071



100*



10.0



0.785



0.650



Positive Control Item



0.030



0.027



0.005



3.8



0.7



0.021



0.029



Test Item



0.159


0.232

0.066



32.9



9.4



0.289



0.248



OD = Optical Density


SD = Standard deviation


* = The mean viability of the negative control tissues is set at 100%.


Negative control: Phosphate buffered saline (PBS)


Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)

Interpretation of results:
other: Under the conditions of the study, the test item demonstrated the ability to cause a positive skin irritation response. However this study alone does not allow the conclusion on whether the test item is EU CLP/UN GHS Category 1 or Category 2.
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item demonstrated the ability to cause a positive skin irritation response. The following classification criteria apply:
EU CLP Category 1 - Code H314; Statement “Causes severe skin burns and eye damage” or EU CLP Category 2 - Code H315; Statement “Causes Skin Irritation”
UN GHS Category 1 or 2
This study alone does not allow the conclusion on whether the test item is EU CLP/UN GHS Category 1 or Category 2.
Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 10 μL of the undiluted test item was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μL PBS (negative control) and 3 tissues with 10 μL 5% SDS (positive control), respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 3.8% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 32.9%. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 18%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was below 50% after 15 minutes treatment the test results are considered as positive. Under the conditions of this study the test item demonstrated the ability to cause a skin irritation response. However this study alone does not allow the conclusion on whether the test item is EU CLP/UN GHS category 1 or 2.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Feb 2020 to 14 Sept 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
There is no description of the morphology of the RhE model neither by the skin model supplier nor the laboratory. However this deviation to the guideline is considered acceptable as all the other parameters are within the QC acceptance criteria.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
18 June 2019
Deviations:
yes
Remarks:
There is no description of the morphology of the RhE model neither by the skin model supplier nor the laboratory. However this deviation to the guideline is considered acceptable as all the other parameters are within the QC acceptance criteria.
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 BIS : In Vitro Skin Corrosion: Human Skin Model Test
Version / remarks:
18 december 2006
Deviations:
yes
Remarks:
There is no description of the morphology of the RhE model neither by the skin model supplier nor the laboratory. However this deviation to the guideline is considered acceptable as all the other parameters are within the QC acceptance criteria.
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: single donor, keratinocyte strain 00267
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (0.63 cm²), supplier MatTek In Vitro Life Sciences Laboratories. (EPI-200)
- Tissue batch number(s): Lot no.: 30848
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 18 Feb 2020 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: 19 Feb 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C; 5 % CO2
- Temperature of post-treatment incubation (if applicable): 37 °C, 5 % CO2; for 3 hours with MTT. For further information see below.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: A 1.0 mg/mL MTT solution was prepared from a MatTek MTT-100 kit immediately prior to usage.
- Incubation time: 3h
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: Without reference filter
- Filter bandwidth: 10 nm
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT SC assay (4h, n=3) performed by the RhE supplier shows an OD of 2.001 +/- 0.08, which is within the acceptance criteria (OD btw 1.0 and 3.0)
- Barrier function: ET-50 assay (MTT assay with 100 µL 1% Triton X-100, 4 time-points, n=3) performed by the RhE supplier showed ET50 = 4.8 hrs within the acceptance criteria (4.77-8.72 hrs)
- Morphology: not reported
- Contamination: No contamination detected
- Reproducibility: Historical control data are available in the reports: data collated from July 2019 till February 2020 and show good reproducibility over time (n=23) of the EpiDerm Skin Model from Mat Tek supplier.
Mean OD negative control, 3 min = 1.87 +/- 0.293 (range 1.416-2.273)
Mean OD negative control, 1h = 1.92 +/- 0.270 (range 1.336-2.362)
Mean OD positive control, 3 min = 0.09 +/- 0.014 (range 0.063-0.11)
Mean OD positive control, 1h = 0.07 +/- 0.008 (range 0.059-0.088)

NUMBER OF REPLICATE TISSUES: Two tissues for each exposure time and exposure condition (test item, negative or positive control, killed tissue)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and non-viable, freeze-killed, tissues.
Freeze-killed tissues possess no metabolic activity but absorb and bind the test item like viable tissues.
Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12-well plate and storing in a freezer (−35 to −10 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour in an incubator at 37 °C, 5% CO2.
In addition to the normal test procedure, the MTT reducing test item was applied to two freeze-killed tissues per exposure period. In addition, two freeze-killed tissues per exposure period remained untreated. The untreated freeze-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

A test item may interfere with the MTT endpoint if it is colored or if it becomes colored when in wet or aqueous conditions. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 μL of test item was added to 300 μL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1) for exposure time 3 minutes and exposure time 1-hour

PREDICTION MODEL:
- The test substance is considered to be corrosive to skin if : the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if: the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Cut off points in accordance with OECD TG 431 and the GHS and CLP Classification systems.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: Not applicable.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test substance at exposure times 3-minutes and/or 1-hour with 3-hours incubation. Where necessary, direct MTT reduction, colour interference was completed.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): Not applicable (sterile distilled water)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N KOH(aq) (pre-diluted), used as supplied
Duration of treatment / exposure:
Observations are made 3-minutes and 1-hour post-test item application
Duration of post-treatment incubation (if applicable):
The DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol over night at room temperature.
Number of replicates:
2/exposure period and control groups
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minute exposure
Run / experiment:
mean (n=2)
Value:
97.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour exposure
Run / experiment:
mean (n=2)
Value:
102.8
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: None reported.
- Direct-MTT reduction: The MTT solution containing the test item turned blue. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using non-viable, freeze-killed, tissues was performed. The results of the freeze-killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
- Colour interference with MTT: The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has previously demonstrated technical proficiency. Also see historic control data.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data.

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test item


 


 



























































































 

 


 


Tissue



 


Exposure Period



Mean OD570 of individual


tissues



Corrected Mean OD570 (tvt-[tkt-


ukt])



Mean OD570 of


duplicate


tissues



 


Standard Deviation



 


Coefficient of Variation (%)



Relative Mean Viability


(%)



 


 


Negative Control



 


3 Minutes



2.243



 



 


2.225



 


0.026



 


1.2



 


 


 


100*



2.206



 


60 Minutes



2.001



 



 


2.133



 


0.186



 


8.7



2.264



 


 


Positive Control



 


3 Minutes



0.094



 



 


0.082



 


0.018



 


na



 


3.7



0.069



 


60 Minutes



0.100



 



 


0.084



 


0.023



 


na



 


3.9



0.067



 


 


 


Test Item



 


3 Minutes



2.153



2.135



 


2.178



 


0.060



 


2.8



 


97.9



2.238



2.220



 


60 Minutes



2.375



2.314



 


2.192



 


0.173



 


7.9



 


102.8



2.131



2.070



OD = Optical density
tvt = Treated viable tissues
tkt = Treated killed tissues
ukt = Untreated killed tissues
* = The mean % viability of the negative control tissue is set at 100%
na = Not applicable


3 minute exposure corrected mean OD570:


x̅ (0.237 + 0.148) = 0.193 (tkt) – x̅ (0.163 + 0.186) = 0.175 (ukt) = 0.018


 


60 minute exposure corrected mean OD570:


x̅ (0.188 + 0.258) = 0.223 (tkt) – x̅ (0.162 + 0.161) = 0.162 (ukt) = 0.061


 


The positive control had a mean relative tissue viability of 73.9% after the 1-hour exposure. The absolute mean OD570 of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤ 30%, indicating that the test system functioned properly. All assay acceptance criteria were met.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is not considered to be corrosive to the skin.
Executive summary:

The study was performed according to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three dimensional epidermal model (EpiDerm). The test was performed by exposing 2 tissues to 50µL of undiluted test item ST 08 C 19 for each time exposure conditions (3min and 1h) together with a negative control (50 μL sterile water) and positive control (50 μL 8N KOH).


The test item was found to directly reduce MTT and therefore additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. 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 of 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-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).


The positive control had a mean relative tissue viability of 3.9% after the 1-hour exposure. The absolute mean OD570 of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20-100% viability the Coefficient of Variation between tissue replicates was ≤ 30%, indicating that the test system functioned properly. All assay acceptance criteria were met.


Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean viabilities for each treatment group, corrected for direct MTT reduction, obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 97.9% and 102.8%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive. Under the conditions of this study, the test item ST 08 C 19 is not considered to be corrosive to the skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22-07-2019 to 22-07-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
other:
Remarks:
Spring Chickens (Gallus Gallus e.g. Ross 308 Broiler)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline. Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
- Time interval prior to initiating testing:
- Indication of any existing defects or lesions in ocular tissue samples: None.
- Indication of any antibiotics used: None reported.
- Selection and preparation of corneas: Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5. Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.
- Quality check of the isolated corneas: Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.
Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.
After the approval process the eyes were incubated for approximately 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.03 ml of test material, undiluted
Duration of treatment / exposure:
The test item remained for 10 seconds in place and then was rinsed from the eye using 20 ml of isotonic saline.
Controls (negative and positive control items) were similarly applied to the cornea in the negative and positive control groups respectively.
Observation period (in vivo):
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.
Number of animals or in vitro replicates:
The test item and positive control item groups consisted of three eyes and the negative control item group consisted of two eyes.
Details on study design:
Selection and preparation of isolated eyes:
Full details are provided in the full study report. Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.
Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline.
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.
Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.
Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.
Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes. After the approval process the eyes were incubated for approximately 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.
Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. 0.03 mL of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.
0.03 mL of the positive control item, Benzalkonium chloride (5% v/v), was similarly applied to the cornea of each positive control eye and 0.03 mL of the negative control item was applied to the cornea of each negative control eye.

Equilibration and baseline recordings: Taken at zero after 45 minutes incubation prior exposure.

Number of replicates: Triplicate for test item and positive control and duplicate for negative controls.

Application dose and exposure time: Application dose: 0.03 ml; Exposure time: 10 seconds.

Observation period: prior to treatment at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

Removal of test substance: Test item remained for 10 seconds and then rinsed with 20ml of isotonic saline.

Methods for measured endpoints:
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.
After the final examinations at the 240 minute time point, the eyes were preserved in neutral buffered formalin for possible histopathology. Eyes were retained until finalization of the final report.
Percentage corneal swelling was assessed from corneal thickness measurements.
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item.
Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.
Once each endpoint had been established, ICE classes were determined based on a predetermined range in accordance with the criteria given in OECD TG 438. The overall in vitro irritancy classification of the test item was determined by using all the classification information and criteria given in OECD TG 438 and the UN GHS classification referenced in the guideline. Full details are provided in the full study report.
The test was considered acceptable as the concurrent negative or vehicle/solvent items and the concurrent positive controls were within the historic control data range and/or identified as GHS non classified and GHS category 1, respectively.
Irritation parameter:
percent corneal swelling
Run / experiment:
mean (n=3)
Value:
1.55
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
mean (n=3)
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
mean (n=3)
Value:
0.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive and negative controls were within the historic control range (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Table 1.- Ocular reactions



























































Test Item
Maximal mean score for corneal opacity0,8ICE Class II
Mean score of Fluorescein Retention0,5ICE Class II
Maximal mean corneal swelling compared to time zero1,55%ICE Class II
Positive Control Item
Maximal mean score for corneal opacity3,3ICE Class IV
Mean score of Fluorescein Retention3ICE Class IV
Maximal mean corneal swelling compared to time zero39,06%ICE Class IV
Negative Control Item
Maximal mean score for corneal opacity0,3ICE Class I
Mean score of Fluorescein Retention0ICE Class I
Maximal mean corneal swelling compared to time zero1,63%ICE Class I

Corneal opacity scores


Maximum score was 1 in the test item group. Maximum score was 0.5 in the negative control. In the positive control group the Maximum score was 4.


No corneal effects were noted in one negative control treated eye during the study period.


Very faint opacity was noted in one negative control treated eye.


Severe corneal opacity was noted in two positive control treated eyes.


Complete corneal opacity was noted in one positive control treated eye. 


Very faint opacity was noted in one test item treated eye.


Scattered or diffused areas of opacity were noted in two test item treated eyes.


No morphological effects were noted in the test item, positive or negative control item treated eyes.


Fluorescein Retention scores


Maximum score was 0.5 in the test item group. Maximum score was 3 in the positive control group, and 0.0 in the negative control. 


No fluorescein retention was noted in the negative control treated eyes during the study period.


Confluent large areas of the cornea retaining fluorescein was noted in all positive control treated eyes. 


Very minor single cell staining was noted in the test item treated eyes.


 


Table 2.- Individual scoring - Test Item
































































































































































































End PointEye NumberTime
(after decontamination)
030 minutes75 minutes120 minutes180 minutes240 minutes
minutes
Corneal Opacity3A0.50.50.5111
6A0.50.51111
8A0000.50.50.5
Mean0.30.30.50.80.80.8
ICE ClassII
Fluorescein Retention3A 0.5    
6A 0.5    
8A 0.5    
Mean 0.5    
ICE ClassI
Corneal Thickness 3A0.660.640.680.660.640.65
6A0.630.610.660.640.620.64
8A0.650.610.630.640.590.59
Mean0.650.620.660.650.620.63
Mean Corneal Swelling (%) -4.121.550.00-4.64-3.09
ICE ClassI
Epithelium Condition3A NNNNN
6A NNNNN
8A NNNNN
ICE Classes Combined:2 x I, 1 x II
Classification:No Category 
Interpretation of results:
GHS criteria not met
Conclusions:
Following assessment of the data for all endpoints the Test Item was considered unlikely to have the potential to cause corrosivity/severe irritancy in vivo (UN GHS No Category and/or EU CLP No Category).
Executive summary:

The study was performed according to OECD TG 438 in accordance with GLP to evaluate possible corrosivity or severe irritancy potential of the Test Item as measured by its ability to induce toxicity in an enucleated chiken eye. 


Method 


0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with negative control item.


Results


Maximal ocular irritation observations recorded for the test item treated eyes were as follows:


























































Mean Corneal OpacityMean Fluorescein RetentionMean Corneal Thickness compared to time zero % (ICE class)Combination of the 3 Endpoints
(ICE class)(ICE class)3075120180240
  minsminsminsminsmins
0.80.5-4.121.550.00-4.64-3.09n/a
(II)(I)(I)(I)(I)(I)(I)
  (I)2 x I, 1 x II
Classification:No Category 

Conclusion


Following assessment of the data for all endpoints the test item was considered unlikely to have the potential to cause ocular corrosivity/severe irritancy in vivo (UN GHS No Category and/or EU CLP No Category).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28-1-2020 to 30-1-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: Human-derived epidermal keratinocyte tissues
Strain:
not specified
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: The test method is indicated as a validated test method recommended by OECD and/or recognised internationally for the sequential testing strategy for the assessment of ocular irritation.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ (OCL-200-EIT MatTek Corporation, for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 02 October 2017; Lot: 30642, Certificate of Analysis provided in the full study report).
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): 50 µL of the test item
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable.
- Concentration (if solution): Not applicable.
- Lot/batch no. (if required): Not applicable.
- Purity: Not applicable.
Duration of treatment / exposure:
30 ± 2 minutes at 37 °C
Duration of post- treatment incubation (in vitro):
120 ± 15 minutes at 37°C post treatment / rinsing incubation
- Rinsing consisted of Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed assay medium (room temperature) in a prelabelled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post- Soak).
- Dried culture inserts were then transferred to 6-well plates containing 1.0 mL of assay medium and were post-treatment incubated for 120 ± 15 minutes at 37°C, 5% CO2.
Number of animals or in vitro replicates:
Two tissues were used for each treatment group (test item, negative control and positive control).
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ (MatTek In Vitro Life Science Laboratories, Bratislava - Slovakia, Lot: 30642, Assay Medium Lot Number: 012720ISA, Certificate of Analysis provided in the full study report).
- Doses of test chemical and control substances used: 50 µL of the test item / 50 µL of the negative control (Sterile water) / 50 µL of the positive control (Methyl Acetate).
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Replicate tissues were equilibrated 15 minutes at room temperature in the 24-well shipping containers. Subsequently, the insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for 1 hour in Assay Medium. After 1 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 (16 to 24 hours). After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++ Mg++ free DPBS to mimic the wet condition of the human eye. If the Ca++ Mg++ free DPBS was not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at 37 °C, 5% CO2 for 30 ±2 minutes. The liquid test item was applied undiluted (50 μL) directly on top of two tissues. Test item was spread to match the size of the tissues. Two tissues were treated with 50 μL Sterile water (negative control) and two tissues with 50 μL Methyl Acetate (positive control) respectively. After the exposure period with test item (30 ±2 minutes at 37ºC), the tissues were throughly rinsed with Ca++ Mg++ free DPBS at room temperature to remove residual test item. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of assay medium at room temperature in a pre-labeled 12-well plate for a 12 ±2 minutes immersion incubation (post‐treatment immersion) 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‐treatment immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, the insert was blotted on absorbent paper, and transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium at approximately 37 °C. The tissues were incubated for a period of 120 ±15 minutes at 37 °C, 5% CO2 (post-treatment incubation).
- Justification for the use of a different negative control than ultrapure H2O: Not applicable.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): not applicable.
- Description of any modifications to the test procedure: Not applicable.
- Indication controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Colour interference was checked for possible colour interference before the study was started. For this purpose 50 μL of the test item was added to 1.0 mL of water in a 6-well plate and the mixture was incubated in the dark at 37 ±1 °C in a humidified atmosphere of 5 ±1% CO2 in air for at least 1 hour. Furthermore, 50 μL of the liquid test item was added to 2 mL of isopropanol, the same amount as used for MTT extraction, incubated in 6 well plates, and placed on an orbital plate shaker for 3 hours at room temperature. The absorbance at 570 nm (OD570) was measured using the LabTech LT-4500 microplate reader and LT-com analysis software. The MTT solution containing the test item turned blue. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using non-viable, freeze-killed, tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes. The water and isopropanol solutions were colorless. It was therefore unnecessary to run color correction tissues.
- Number of tissue replicates used per test chemical and controls (positive, negative): Duplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device: 570 nm.
- Description of the method used to quantify MTT formazan: Labtech LT-4500 microplate reader.
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to the OECD TG 492 and GHS system.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Formerly, there were eight of these study types performed at this testing facility prior to the conduct of this study. The historical control values obtained for the negative and positive controls in this study were slightly lower than those obtained in the previous eight studies. However, this occurrence was considered inconsequential since the Historical Control Data is based on a finite number of studies and also the control data obtained for this study was within the OECD TG 492 negative and positive control acceptance ranges (See: Acceptance Criteria) and therefore the study met the criteria for an acceptable test. The current HCD was provided in the full study report.
- Complete supporting information for the specific RhCE tissue construct used: Attached to the full study report.
- Reference to historical data of the RhCE tissue construct: Full historic control data is attached to the full study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The test assay is validated at the test laboratory and this information is in the public domain.
- Positive and negative control means and acceptance ranges based on historical data: See above.
- Acceptable variability between tissue replicates for positive and negative controls: <10%.
- Acceptable variability between tissue replicates for the test chemical: Yes.
Irritation parameter:
mean percent tissue viability 
Run / experiment:
mean (n=2). Positive control.
Value:
11.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
mean percent tissue viability 
Run / experiment:
mean (n=2). Test item.
Value:
106.5
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: None.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test assay is validated at the test laboratory and this information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: See 'details on study design' for further information. The laboratory historical control data is presented in the full study report. Formerly, there were eight of these study types performed at this testing facility prior to the conduct of this study. The historical control values obtained for the negative and positive controls in this study were slightly lower than those obtained in the previous eight studies. However, this occurrence was considered inconsequential since the Historical Control Data is based on a finite number of studies and also the control data obtained for this study was within the OECD TG 492 negative and positive control acceptance ranges (See: Acceptance Criteria) and therefore the study met the criteria for an acceptable test.
















































Item



OD570 of tissues



Mean OD570 of duplicate tissues



Individual tissue viability (%)



Relative mean viability (%)



Difference in viability (%)



Negative Control Item



1.593



1.613



98.8



100*



2.4



1.632



101.2



Positive Control Item



0.200



0.186



12.4



11.5



1.8



0.171



10.6



Test Item



1.832



1.718



113.6



106.5



14.2



1.604



99.4



 


OD =  Optical Density


* =          The mean viability of the negative control tissues is set at 100%


 


Acceptability of the assay: 


The relative mean tissue viability for the positive control treated tissues was 11.5% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.
The mean OD570 for the negative control treated tissues was 1.613. The negative control acceptance criterion was therefore satisfied.
The difference in viability between the two relating tissues in each treatment group was <20%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was classified as non-irritant. The following classifications apply:
EU CLP and UN GHS No Category
Executive summary:

Introduction: The purpose of this study was to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular™ Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.


Method: Duplicate tissues were treated with the test item for an exposure period of 30 minutes. At the end of the exposure period each tissue was rinsed before incubating for 120 minutes. The test item was found to directly reduce MTT and therefore additional non-viable, freeze-killed, tissues were incorporated into the testing for correction purposes. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction.
At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).


Results: The results obtained showed that negligible interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results.
The relative mean viability of the test item treated tissues was 106.5%.
Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.


Conclusion: The test item was classified as non-irritant. The following classifications apply: EU CLP and 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

Justification for classification or non-classification

The registered substance is classified as skin irritant category 2 (H315) according to the criteria of the Regulation (EC) 1272/2008 (CLP) and to GHS.


The registered substance is not classified for eye irritancy/severe eye damage according to the criteria of the Regulation (EC) 1272/2008 (CLP) and to GHS.


The OECD 492 guideline test has been conducted to confirm the negative results obtained in the OECD 438 guideline test, as it was a registration requirement in another country.