Registration Dossier

Administrative data

Description of key information

Skin irritation (OECD 439), EpiSkin: not irritating

Eye irritation (OECD 437), BCOP: not corrosive

Eye irritation (OECD 492), Epiocular RhCE: irritating potential

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:
02 - 24 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm, reconstructed three-dimensional human epidermis (EPI-200)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 28697
- Delivery date: 21 May 2019
- Date of initiation of testing: 21 May 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5°C for 35 min in the incubator followed by 25 min at room temperature under the sterile flow
- Temperature of post-treatment incubation: 37 ± 1.5 °C (42 h)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with PBS for at least 15 times in order to remove any residual test material. After rinsing, the inserts were submerged in PBS at least 3 times. Afterwards the inserts were once again rinsed with sterile PBS from the inside and the outside. Excess PBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.673 ± 0.124 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.62 h (acceptance criteria: 4.77-8.72 h).
- Morphology: no visible defects observed
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.
- Reproducibility: Concurrent negative and positive controls fell within historical control data

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce the MTT solution and no colour interference was observed, an additional functional check was not performed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is ≤ 50%.
- The test substance is considered to be non-irritant to skin if the viability after 1 hour exposure is > 50%.

TEST ACCEPTANCE CRITERIA
EpiDermTM (EPI-200-SIT) model
Negative control: Lower acceptance limit ≥ 0.8, Upper acceptance limit ≤ 2.8
Positive control: mean relative tissue viability of the positive control is ≤ 20%
Standard deviations (SD): SD of 3 identical replicates should be ≤ 18
OD values: OD values should not be below historically established boundaries.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg, wetted with 25 μL DPBS

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% in deionised water
Duration of treatment / exposure:
35 min at 37°C and 25 min at room temperature
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
1 h exposure
Value:
94.18
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 visible damage was observed.
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: The test substance did not change the colour, when mixed with deionised water and thus passed the colour interference pre-test. Also it´s intrinsic colour was not intensive.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.780 and 1.890).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 h was ≤ 20% compared to the negative control (4.17%).
- Acceptance criteria met for variability between replicate measurements: Standard deviation of 3 identical replicates was ≤ 18 thus ensuring the validity of the study.

Table 1: Results 1 h exposure

Treatment group

Tissue No.

OD 570 nm

Mean OD

Mean OD (blank corrected)

Rel. Viability [%] Tissue 1, 2, 3*

 Standard Deviation

Mean Rel. Viability [%]**

Blank

 

Well 1

Well 2

Well 3

 

 

 

 

 

 

 

0.037

0.037

0.037

0.037

 

 

 

 

Negative control

1

1.865

1.780

1.832

1.826

1.789

98.260

 

 

2

1.885

1.877

1.890

1.884

1.847

101.470

1.6

100.0

3

1.896

1.841

1.849

1.862

1.825

100.269

 

 

Positive control

1

0.114

0.114

0.115

0.114

0.077

4.256

 

 

2

0.109

0.111

0.113

0.111

0.074

4.060

0.1

4.17

3

0.113

0.115

0.111

0.113

0.076

4.188

 

 

Test item

1

1.623

1.571

1.575

1.590

1.553

85.299

 

 

2

1.946

1.897

1.915

1.919

1.882

103.389

9.0

94.18

3

1.784

1.718

1.733

1.745

1.708

93.838

 

 

* Relative viability [rounded values]

** Mean relative viability [rounded values]

Table 2: Historical Control data

Positive Control; OD at 570 nm after exposition to 5% SDS solution in deionized water (MatTek)

 Negative Control OD at 570 nm DPBS (MatTek)

Tissue Viability [%]

3.99

Mean OD

1.69

Standard Deviation

1.04 % points

Standard Deviation

0.19

Range of Viabilities

2.24% - 6.19%

Range of OD*

1.28 - 2

Mean OD

0.07

 

* should be 0.8 - 2.8 (OECD 439)

or 1.0 - 2.5 (MatTek)

Standard Deviation

0.02

Table 3:In vitro Skin Irritation Assay: OECD 439 Proficiency Data (March 2014, non-GLP) performed at Envigo CRS GmbH the testing facility

Proficiency Substance

Viability [%]

Category

Naphtalene acetic acid

101.7

No Cat.

Isopropanol

85.5

No Cat.

Methyl stearate

91.1

No Cat.

Heptyl butyrate

109.0

No Cat.

Hexyl salicylate

98.1

No Cat.

Cyclamen aldehyde

24.1

Cat. 2

1-Bromohexane

16.3

Cat. 2

Potassium hydroxide (5% aq.)

40.1

Cat. 2

1-Methyl-3-phenyl-1-piperazine

21.6

Cat. 2

Heptanal

20.6

Cat. 2

 

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance did not possess irritating properties towards reconstructed human epidermis tissue in the EpiDerm™ model.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted 9 October 2017
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, Aschaffenburg, Germany
- Characteristics of donor animals: 14 months old
- Storage, temperature and transport conditions of ocular tissue: The isolated bovine eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% [v/v] penicillin/streptomycin.
- Time interval prior to initiating testing: On the test day, fresh eyes were collected from the slaughter house. The corneae were isolated on the same day after delivery of the eyes.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: 1% [v/v] penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) containing HBSS was used for storage in the slaughter-house and for transport.


Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20%

VEHICLE
- Substance: Physiological saline
- Amount applied: 750 µL
- Concentration: 0.9% Natrium chloride in deionised water

POSITIVE SUBSTANCE
- Substance: Benzalkonium chloride
- Concentration: 10% Benzalkonium chloride in 0.9% Natrium chloride solution in deionised water
- Amount applied: 750 µL
Duration of treatment / exposure:
4 h at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
90 ± 5 min at 32 ± 1 °C
Number of animals or in vitro replicates:
triplicates each for treatment and control groups
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
- Dissection of the eyes and treatment: Corneas were carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated corneas.
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws.
- Test medium and temperature conditions used in the cornea holder: The incubation medium consisted of Minimum Essential Medium (MEM) supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS) and prior to use it was pre-warmed to 32 ± 1 °C.
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: Only corneaes with an initial basal opacity value < 7 were used

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Corneas were washed at least 3 times with Minimum Essential Medium (MEM) containing phenol red (or more if phenol red was still discoloured (yellow or purple), or the test item was still visible). Once the medium was free of the test item the corneaes were given a final rinse with MEM containing all supplements without phenol red.
- POST-EXPOSURE INCUBATION: 90 ± 5 min at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP KiT opacitometer (Electro Design, 63-Riom, France)). The change of the opacity value of each treated cornea or of the positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea. The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microtiter plate reader (Versamax® Molecular Devices) at 490 nm (OD490). The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- Calculation IVIS of the negative control: IVIS = opacity value + (15 x OD490 value), Calculation IVIScorrected of the positive control and the test item: IVIScorrected = (opacity value – mean of opacity of negative control) + 15 x (permeability value – mean permeability of the negative control)

DECISION CRITERIA:
Test substance with an IVIS ≥ 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3 ≤ 55 was regarded as no prediction can be made.

ACCEPTABILITY CRITERIA
The test will be acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
A single testing run composed of at least three corneae should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered, as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneae are non-concordant, such that:
- 2 of the 3 corneae give discordant predictions from the mean of all corneae, or,
- 1 of the 3 corneae gives a discordant prediction from the mean of all 3 corneae, and the discordant result is >10 IVIS units from the cut-off threshold of 55.
- If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.


Irritation parameter:
in vitro irritation score
Remarks:
mean out of all 3 eyes
Run / experiment:
4 h
Value:
3.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Test substance
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No macroscopically visible defects were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control gives a mean IVIS of 0.77, the opacity (mean 0.00) and permeability (mean 0.051) values are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control
- Acceptance criteria met for positive control: yes, the positive control gives a mean IVIS of 91.16, that falls within two standard deviations of the current historical mean

Table 2: Results after 240 minutes treatment time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

 

 

Mean

 

Mean

 

 

Negative Control

0

0.00

0.054

0.051

0.81

0.77

0

0.050

0.75

0

0.050

0.75

Positive Control

74.00*

0.404*

80.06

91.16

99.00*

0.243*

102.64

83.00*

0.520*

90.80

Test Item

3.00*

-0.004*

3.00

3.40

3.00*

0.013*

3.19

4.00*

-0.003*

4.00

*corrected values

Interpretation of results:
other: non-corrosive
Conclusions:
Under the conditions of the Bovine Corneal Opacity and Permeability Test (BCOP) the test substance was not identified to induce serious eye damage, but the test substance could also not be identied to not require classification for eye irritation or serious eye damge. Thus, the test substance is considered to be non-corrosive, but no prediction on the irritation potential can be made and further evaluation and/or data generation is required.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 - 27 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
human
Strain:
other: EpiOcular™, reconstructed three-dimensional human corneal epithelium
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Purity: : ≥ 98%
Duration of treatment / exposure:
6 h at 37 ± 1.5°C and 5 ± 0.5% CO2
Duration of post- treatment incubation (in vitro):
Post-exposure immersion: 25 min at room temperature
Post-treatment incubation: 18 h at 37 ± 1.5°C and 5 ± 0.5% CO2
Number of animals or in vitro replicates:
duplicate tissues for each treatment and control group
Details on study design:
- Details of the test procedure used : The EpiOcular ™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.
- RhCE tissue construct used, including batch number : EpiOcular ™ tissue OCL-200, OCL-212, MatTek Corporation, Bratislava, Slovakia, Lot number: 30612
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment.
To test if a test substance directly reduces MTT, 1 mL of a MTT solution (1 mg/mL) including 50 mg of the test substance were incubated for 180 min under standard conditions. 50 µL deionised water in MTT solution were used as negative control. After incubation the change of color was determined. Since the MTT solution colour did not turn blue/purple, the test substance is not presumed to have reduced the MTT. An additional test with freeze-killed tissues did not have to be performed.
The optical pre-experiment (color interference pre-experiment) to investigate the test item’s color change potential in water or isopropanol did not lead to a change in colour.
To test ability of the test substance for colour interefrence, 50 mg of the test substance was added to 1 mL of deionised water and mixed. 1 mL of deionised water was used as control (blank). Both were incubated for 60 min. In parallel, 50 mg of the test substance was added to 2 mL of isopropanol and mixed. A control (2 mL of isopropanol, blank) was run concurrently. Both were incubated for 3 h at room temperature. After incubation the presence of the staining was evaluated by OD measurement.
- Wavelength used for quantifying MTT formazan: 570 nm
- MTT concentration: On the day of the experiment a MTT solution of 1 mg/mL in DMEM was prepared.
- MTT incubation time: Tissues were incubated for 180 min in 300 μl MTT solution. Each tissue were extracted with isopropanol within 2.5 h while shaking at room temperature. Then, the extracts were mixed and two 200 μL aliquots were transferred to a 96-well plate for OD measurement. 200 μL of isopropanol were added to the wells designated as blanks for 96-well plate.
- Description of the method used to quantify MTT formazan : The absorbance (OD570) was measured with a plate reader (Versamax® Molecular Devices, Software Softmax Pro Enterprise, version 4.7.1).
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : The test substance is considered to be not irritating to eye if the tissue viability after 6 h exposure, 25 min post-exposure immersion and 180 min post-exposure incubation is >60% relative to the negative control treated tissue.
- Complete supporting information for the specific RhCE tissue construct used :
Viability: The quality ofthe tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.758 ± 0.071 (acceptance criteria: 1.1 - 3.0).
Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 23.65 min (acceptance criteria: 12.2-37.5 min).
Contamination: The tissues were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.
- Reference to historical data of the RhCE tissue construct : See Table 1 in the section "Any other information on materials and methods incl. tables"
- Acceptable variability between tissue replicates for positive and negative controls : The results are acceptable if the negative control OD is >0.8 and <2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.
- Acceptable variability between tissue replicates for the test chemical: The results are acceptable if the difference of viability between the two relating tissues of a single test item is < 20% in the same run.

Irritation parameter:
other:
Remarks:
% tissue viability / mean value of 2 tissues
Run / experiment:
6 h exposure
Value:
2.35
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes proficiency was demonstrated (correct prediction of the irritation potential of the proficiency chemicals, meeting all acceptance criteria for positive and negative controls defined by the OECD 492).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the negative control was > 0.8 and < 2.5 (mean OD 1.974).
- Acceptance criteria met for positive control: The mean relative viability of the positive control was < 50% compared to the negative control (28.7%).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) of viability between tissue replicates is < 20% (values between 0.17% and 9.25%).

Table 2: Results after treatment with test item and the controls for 6 h

Test Group

Tissue No.

Well 1 [OD570]

Well 2 [OD570]

Mean [OD570] (Well 1 and

well 2)

Mean [OD570] blank corr. (Well 1 and

well 2)

Mean [OD570] of T1 and T2

Tissue viabil. [%]

rel. viabil. of T1 and T2

Diff. of viabil. between T1 and T2 [p.p.]

Blank

 

0.037

0.036

0.037

 

 

 

 

 

Negative control

1

2.112

2.090

2.101

2.065

1.974

100.00

104.6

9.25

2

1.909

1.929

1.919

1.882

95.4

Positive control

1

0.574

0.596

0.585

0.548

0.566

28.70

27.8

1.82

2

0.619

0.622

0.621

0.584

29.6

Test item

1

0.080

0.083

0.081

0.045

0.046

2.35

2.3

0.17

2

0.085

0.085

0.085

0.048

2.4

Interpretation of results:
other: irritating potential
Conclusions:
After treatment with the test item, the mean value of relative tissue viability was 2.35%. This value is below the threshold for eye irritation potential (≤ 60%). Under the conditions of the conducted test, the test substance is considered to possess an irritating potential towards human cornea in the EpiOcular™ model but the result is not conclusive with respect to classification of the test substance as eye irritant (Eye Irritant Cat. 2) or serious eye damage (Eye Damage Cat. 1) and therefore requires further evaluation and/or data generation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate was tested for its skin irritation potential in the EpiDermTM human skin model according to OECD Guideline 439 and in compliance with GLP (2019).

Preliminary tests for direct MTT reduction and colour interference of the test substance did not indicate the necessity of additional tests with freeze-killed or viable tissues.

25 mg of the test item, 30µL of the negative control (DPBS) and 30 µL of the positive control (5% SDS) were applied to each three tissues of reconstructed human epidermis for 1 h (35 min at 37°C and 25 min at room temperature).

After the treatment the test item and control substances were washed from the skin surface with phosphate-buffered saline followed by a 42 h post-incubation period. For determining alterations in cell viability, MTT reduction assays were performed. The tissue viability (mean of 3 tissues) for the test item was 94.18% after 1 h. Since the evaluation criteria for non-irritants (> 50% viability after 1 h) were met, the test substance was considered to be non-irritant to skin.

Acceptance criteria for positive control, negative control and variability between tissue replicates were met. After treatment with the positive control substance SDS the mean viability of tissue replicates was reduced to 4.17% after 1 h and therefore within the required range of ≤ 20%. The mean OD of the tissue replicates treated with the negative control was between ≥ 0.8 and ≤ 2.8 for the 1 h treatment period, thus assuring the quality of the tissues. Furthermore, the standard deviation of 3 identical replicates was ≤ 18.

In conclusion, Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate is considered to be not irritant to skin.

 

Eye irritation

Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate was tested for its eye hazard and damage potential in the Bovine Corneal Opacity and Permeability (BCOP) assay according to OECD Guideline 437 and in compliance with GLP (2019).

Fresh isolated bovine eyes were collected at the day of the experiment. Corneas were dissected with a 2 mm rim of sclera and were mounted in cornea holders (3 corneas per group).

After an equilibration period and a first opacity measurement, 750 µL of the test substance solution (20% in physiological saline) was applied to the cornea and incubated for 4 h at 32± 1 °C (closed-chamber method). The positive control 10% (w/v) benzalkonium chloride and the negative control, saline (0.9% (w/v) NaCl in deionised water) were applied at the same volume. After treatment, the corneas were washed with Minimum Essential Medium containing phenol red for at least three times. Subsequently, the corneal opacity was determined again by the amount of light transmission through the cornea via an opacitometer. Permeability of the corneas were determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation for 90 min at 32 ± 1 °C. measured. Based on opacity and permeability, the In Vitro Irritancy Score (IVIS) was calculated. Relative to the negative control, the test substance caused an increase of the corneal opacity. The calculated mean IVIS was 3.40 (threshold for serious eye damage: IVIS > 55, threshold for no category: IVIS ≤ 3). Since the IVIS value was slightly above the threshold of 3 but less than ≤ 55 no prediction can be made regarding for the damage potential of the test substance to the eye. For the negative control, neither an increase of opacity nor permeability of the corneae was observed (mean IVIS 0.77). The positive control showed significant opacity and distinctive permeability of the corneae (mean IVIS 91.16) corresponding to a classification as serious eye damaging (EU CLP/UN GHS (Category 1)). In conclusion, for the test substance with an IVIS > 3 ≤ 55 no prediction can be made regarding its irritant property. In conclusion, since the threshold for identification of substances causing serious eye damage was not met, the test substance in considered to be non- corrosive.

Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate was tested with the Reconstructed human Cornea-like Epithelium (RhCE) method (EpiocularTM) which allows the identification of test substance that do not require classification and labelling for eye irritation or serious eye damageaccording to OECD Guideline 492 and in compliance with GLP (2019).

50 mg of the test item or 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were applied to each duplicate tissue for 6 h. After an 25 min immersion and 18 h post-treatment incubation, the tissues were incubated with MTT solution for 180 min. Subsequently, MTT reduction was determined by the measurement of optical density (OD570nm) using a microplate reader. In preliminary experiments, the test substance did not prove to be a MTT reducer and no color interference was observed. Therefore, additional tests with freeze-killed tissues or viable tissues did not have to be performed.

The mean relative viability value of test substance treated tissues was 2.35% compared to the mean value of the negative control. This value is below the threshold (≤ 60%) for substances not requiring classification for eye hazard potential.

All acceptability criteria were fulfilled. The mean OD of the tissue replicates treated with the negative control was 1.974 and therefore well within the acceptable range of > 0.8 and < 2.5. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of relative viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

In conclusion, Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate reduced viability below the threshold of 60%, for substances not requiring classification for eye hazard potential. Therefore, the test substance has eye irritation or damaging potential. The specific hazard class cannot be determined with this test method.

 

Taken together, based on the results of the BCOP assay and the Reconstructed human Cornea-like Epithelium (RhCE) assay an eye irritation potential (H319) is concluded for Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate. This conclusion can be drawn because the BCOP assay showed that the test substance is not corrosive to the eye and the RhCE assay indicated an eye irritating or damaging potential. As the BCOP assay excluded an eye damaging potential the test substance is considered to be eye irritating.

Justification for classification or non-classification

The available data on skin irritation/corrosion of Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification. The available data on eye irritation of Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate meet the criteria for classification as Eye Irrit. 2 ‘Causes serious eye irritation (H319)’ according to Regulation (EC) No 1272/2008.