[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

no

Test guideline 2

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

1

Reference substance name:

Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethy-1H-pyrazol-1-yl) succinate

Molecular formula:

C9H10K2N2O4

IUPAC Name:

Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethy-1H-pyrazol-1-yl) succinate

In vitro test system

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

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.