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Administrative data

Description of key information

Skin irritation (EpiSkin, OECD TG 439), Wingenroth 2021: not irritant


Eye irritation (EpiOcular, OECD TG 492); Leidenfrost 2021: irritant


Eye corrosion (BCOP, OECD TG 437); Leidenfrost 2021: not corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept-Nov 2021
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)
Version / remarks:
2021
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)
Specific details on test material used for the study:
-Purity (HPLC area %) 100
-Test item was used undiluted.
-For the solid test item 30 µl of 0.9% NaCl were used to moisten and ensure good contact with the epide1mis surface.
Test system:
human skin model
Source species:
human
Justification for test system used:
commercially available test method
Vehicle:
other: 0.9% NaCl
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (Henkel AG & Co, KGaA, Düsseldorf))

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2
gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION
- The optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA
- The mean optical density (OD) values obtained with the test item were used to calculate the
percentage of viability relative to the negative control, which is set at 100 %.
- According to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline
Duration of treatment / exposure:
20 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
cell viability, after 20 min [%]
Value:
100.58
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 - according to the results of the pre-check
- Colour interference with MTT: No - according to the results of the pre-check

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

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

Table 1: Tabular summary of the results    


 

































Sample No.Test item OD mean * Std Dev % Viability 
 1 - 3

 Negative control NaCl 0.9 %



1.83



0.02



100.00



 4 - 6



 Positive control SDS 5 % 



0.02



0.00



1.31



 7 -9



test item (DFPP MALAT)



1.84



0.01



100.58



 


* 6 values

Interpretation of results:
GHS criteria not met
Conclusions:
not irritant, no classification warranted
Executive summary:

A study was performed for the assessment of the skin irritancy of the test item DFPP MALAT with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.
The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.
The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.
Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%.
The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.
The following value of cell viability was recorded for the test item: 101 % (rounded).
In conclusion the results of the assay used show no skin initant properties of the test item DFPP MALAT and thus, the test item requires no classification according to CLP.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct - Nov 2021
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)
Specific details on test material used for the study:
-Purity (HPLC area %) 100
- used as solid
Species:
other: reconstructed human cornea-like epithelium (RhCE)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
JUSTIFICATION OF THE TEST METHOD AND CONSIDERATIONS REGARDING APPLICABILITY :
The EpiOcular™ eye irritation test (EIT) follows international OECD test guidelines. The EIT measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation).

DESCRIPTION OF THE CELL SYSTEM USED, INCL. CERTIFICATE OF AUTHENTICITY AND THE MYCOPLASMA STATUS OF THE CELL LINE :
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The model is standardized and commercially available. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier (MatTek Corporation, Slovakia).

ENVIRONMENTAL CONDITIONS :
The environmental conditions in the incubator were standardized as follows:
Incubator temperature: 37 +/- 2° C
CO2 gas concentration: 5 %
Humidity: 95 %
All incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode, Germany).
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 mg per tissue insert in duplicate
- Concentration (if solution): neat test item

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 μLper tissue insert in duplicate
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
tissue inserts were used in duplicate for test item, negative and positive control
Details on study design:
RhCE TISSUE CONSTRUCT USED, INCLUDING BATCH NUMBER :
EpiOcular RhCE tissue supplied by MatTek Corporation,

PRE-CHECK FOR POTENTIAL OPTICAL INTERFERENCES OF THE TEST ITEM :
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT-reduction of the test item
In case of a direct MTT-reduction of the test item a killed tissue control (inserts, which were killed by freezing) was used in the main assay.
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control was used in the main assay.
The evaluation criteria for the pre-check were:
- For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted.
- For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control must be conducted.

PROCEDURE FOR KILLED CONTROL OR COLOR CONTROL :
Killed control and color control not required.

DESCRIPTION OF THE METHOD USED TO QUANTIFY MTT FORMAZAN :
For viability testing the inserts were placed in new plates containing MTT solution (1 mg/ml in Maintenance medium at 37°C). The tissues were incubated for 180 ± 10 min. under standard culture conditions. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 μl). Data acquisition and evaluation were performed with the software "Gen5" (Bio-Tek).

ASSAY ACCEPTANCE CRITERIA :
The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.8
- mean relative viability of the positive control (PC) is < 50 % (relative to negative
control)
- the difference of viability between the two replicates is < 20 %.
Irritation parameter:
mean percent tissue viability 
Value:
2.92
Vehicle controls validity:
valid
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 - according to the results of the pre-check
- Colour interference with MTT: No - according to the results of the pre-check

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS :
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

 































 



Final Cell viability



OD mean (n=4)



Compound



[%]


 

DFPP MALAT


2.92

0.054



Positive control


22.470.418

Negative control


1001.861

 

Conclusions:
irritating properties; further tests warranted
Executive summary:

An in vitro study for assessing ocular irritation properties of the test item DFPP MALAT was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™. The model used is standardized and commercially available.
The EpiOcular™ Eye Irritation Test (EIT) was conducted in accordance with OECD 492.
The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period.
Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.
The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model.
The final mean percent tissue viability recorded for the test item is 3% (rounded).
According to the results of this study the test item DFPP MALAT was identified as  irritant to the eyes and further test are required for classification according to CLP.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2020
GLP compliance:
yes
Specific details on test material used for the study:
Purity: 100%
used as solid
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Number of animals: n/a
- Characteristics of donor animals (e.g. age, sex, weight): not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport: 1 L containers with 500 mL HSS and 1 % penicillin / streptomycin solution, transport of the containers in coolers on ice
- Indication of any existing defects or lesions in ocular tissue samples: Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter. Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.
- Selection and preparation of corneas: On the next day the containers with the eyes were transferred in an incubator at 32 °C (± 1 °C) for about 2 hours. For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed in a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour into the incubator at 32 °C (± 1 °C).
- Quality check of the isolated corneas: After approximately 1 hour, the MEM medium was aspirated, and the chambers were filled with fresh MEM medium. For each cornea the reference opacity value was measured then.
Vehicle:
physiological saline
Remarks:
The test item was suspended with a glass rod, a whirl mixer and shaking shortly prior to application in isotonic saline solution to achieve a concentration of 20 % (w/v). The preparation was visually described as suspension.
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
4 h
Number of animals or in vitro replicates:
three corneae each treatment and /or control
Details on study design:
NUMBER OF REPLICATES: three

NEGATIVE CONTROL USED: yes physiological saline

SOLVENT CONTROL USED (if applicable) yes physiological saline

POSITIVE CONTROL USED yes imidazole 20% solution

APPLICATION DOSE AND EXPOSURE TIME 750 µL of a 20% suspension (w/v)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch).
- Corneal permeability: The medium in anterior chamber of each holder was replaced by 1ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes.
After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 µL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates.
The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader (Bio-Tek EL 808, Software Gen5).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Same decision citreria used as specified in the guideline.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
-7.6
Vehicle controls validity:
valid
Remarks:
-1.7
Positive controls validity:
valid
Remarks:
139.7
Irritation parameter:
other: permeability value
Run / experiment:
mean
Value:
-0.002
Vehicle controls validity:
valid
Remarks:
0.049
Positive controls validity:
valid
Remarks:
1.193
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
7.6
Vehicle controls validity:
valid
Remarks:
-2.4
Positive controls validity:
valid
Remarks:
121.8

Opacity score











































































































 



Cornea No.



Initial opacity



final opacity



Opacity change



Mean



SD



 



Negative control isotonic saline solution



1



7.9



6.4



-1.5



 



 



2



7.4



4.5



-2.9



-2.4 



0.8



3      



      7.2     



      4.4     



      -2.9    



      



     



 



Cornea No.



Initial opacity



final opacity



Opacity change



Corrected opacity change



Mean



SD



Positive control Imidazole



4



7.0



199.9



192.9



195.3



 



 



5



6.6



108.6



102.0



104.4



121.8



66.5



6



 6.5



   69.8  



   63.3  



       65.7      



     



 



Test item



7



6.4



8.1



1.7



4.2



 



 



8



6.3



14.2



7.9



10.3



7.6



3.1



9



 6.1



   12.0  



    5.9   



        8.3       



 



 



 


Permeability score





















































































 



Cornea No.



Mean OD490



Mean



SD



 



Negative control isotonic saline solution



1



0.050



 



 



2



0.046



0.049



0.002



3



0.051



 



 



 



Cornea No.



Mean OD490



Mean corrected OD490



Mean



SD



Positive control Imidazole



4



1.111



1.062



 



 


5

1.538



1.490



1.193



0.257



6



1.076



1.028



 



 



Test item



7



0.046



-0.002



 



 



8



0.050



0.002



-0.002



0.003



9



0.044



-0.005



 



 



 


Tabular In vitro irritancy score
































































































 



Cornea No.



Opacity per cornea



Permeability per cornea



per cornea



IVIS per group



mean



SD



Negative control isotonic saline solution



1



-1.5



0.050



-0.7



 



 



2



-2.9



0.046



-2.3



-1.7



0.8



3



-2.9



0.051



-2.1



 



 



Positive control Imidazole



4



195.3



1.062



211.2



 



 



5



104.4



1.490



126.8



139.7



66.0



6



65.7



1.028



81.1



 



 



Test item



7



4.2



-0.002



4.1



 



 



8



10.3



0.002



10.3



7.6



3.1



9



8.3



-0.005



8.3



 



 



 



 


 


 


 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on OECD TG 437 and the experimental conditions reported the test item is not classified as seriously damaging the eye (no Category).
Executive summary:

This in vitro study was performed to assess them corneal irritation and damage potential of DFPP MALAT by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 2020.


 


The corneae were incubated with the test substance and controls for 4 h. After exposure the corneae were rinsed with phenol red containing MEM. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean corrected OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.


A 20% dilution of the test substance in isotonic saline solution caused no increase of the corneal opacity and permeability. 


The positive control (Imidazole) increased the opacity and permeability of the corneae in this experiment.


With the negative control (isotonic saline solution) neither an increase of opacity nor permeability of the corneae could be observed in the experiment.


Since the mean in vitro irritancy score of the test substance was 7.6, DFPP MALAT is not considered to be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the study results no classification is warranted for skin irritation and classification Cat. 2 (H319) is warranted for eye irritation according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I.