[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:

09 February - 13 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Harpin-αß protein

IUPAC Name:

Harpin-αß protein

Test material form:

liquid

Details on test material:

The test material is identical to the registered substance, 'Cell Free Harpin Extract of Harpinαβ produced by fermentation'.

Specific details on test material used for the study:

TEST MATERIAL
- Lot No. of test material: R134-2
- Expiration date of the lot: 17 July 2018
- Appearance: Tan, light brown liquid
- Active components: Harpin-ab proteins 0.4% in aqueous solution
- Storage: <-70°C protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstituted three-dimensional human skin model EpiDerm™ (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- Tissue batch number(s): Lot no. 25882
- Date of initiation of testing: 14 February 2018 (pre-experimental starting date)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The plates were incubated in a humidified incubator at 37 ± 1°C, 5.0% CO2 for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation: Once the inserts had been washed, they were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well. The plates were then post-incubated at 37 ± 1°C, 5% CO2, humidified to 95% for 24 ± 2 h. Following this incubation, the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for an additional 18 ± 2 h.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface. The inserts were then completely submerged 3 times in 150 mL DPBS and shaken to remove the rest of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS.
- Observable damage in the tissue due to washing: None specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a 1 mg/mL concentration of MTT medium was used per well.
- Incubation time: 3 h ± 5 min at 37 ± 1°C, 5% CO2, humidified to 95%
- Extraction: After the MTT incubation period, the tissues were rinsed 3 times with DPBS and dried. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol and sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 hours with gentle shaking on a plate shaker.
- Spectrophotometer: Plate spectrophotometer
- Wavelength: 570 nm
- Filter bandwidth: Maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC assay, 4 hours, n=3; OD(540 - 570 nm) = 1.52 ± 0.086 = PASS
- Barrier function: ET-50 assay, 100 µL 1% Triton X-100, 4 timepoints, n=3, MTT assay; ET50 = 7.27 hous = PASS
- Morphology: No data
- Contamination: Long term antibiotic and antimycotic free culture; Sterile = PASS
- Reproducibility: No data

NUMBER OF REPLICATE TISSUES: The test was performed on a total of 3 tissues per dose group.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability is greater than or equal to 50% after exposure and post-treatment incubation.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: The test item was applied undiluted. 30 µL (47 µL/cm2) of the test item was dispensed directly atop the EpiDerm™ tissue. The test item was gently spread to match the size of the tissue using a bulb headed Pasteur pipette.
- Concentration: 47 µL/cm2

VEHICLE
- None

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL

Duration of treatment / exposure:

60 ± 1 minutes

Duration of post-treatment incubation (if applicable):

24 ± 2 h and a further 18 ± 2 h (42 h in total)

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Average

Value:

97.3

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:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was >50% (97.3%) after 60 minutes treatment and 42 hour post-incubation.

- OTHER EFFECTS:
- Visible damage on test system: None specified
- Direct-MTT reduction: The mixture of 30 µL test item per 1Ml MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/ purple. Therefore, NSMTT (non-specific reduction of MTT) was 0%.
- Colour interference with MTT: The mixture of 30 µL of the test item per 300 µL distilled water and 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC (non-specific colour) was 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (mean absolute OD570 nm was 2.002)
- Acceptance criteria met for positive control: Yes (mean relative viability was 3.0%)
- Acceptance criteria met for variability between replicate measurements: Yes (standard deviation variability was 0.1% - 9.8%)
- Range of historical values: See Table 2 - Historical data were generated from 2015 to 2017

Any other information on results incl. tables

Table 1: Results of the test item

	Negative control			Positive control					Test item
Tissue	1	2	3	1		2		3	1		2		3
Mean OD570of the duplicates (blank corrected)	2.014	1.743	2.117	0.058		0.060		0.056	1.956		1.847		1.912
Total mean OD570of 3 replicate tissues (blank corrected)	1.958*			0.058					1.905
SD OD570	0.193			0.002					0.055
Relative tissue viability (%)	102.9	89.0	108.1	3.0	3.1		2.9		99.9	94.3		97.6
Mean relative tissue viability (%)	100.0			3.0**					97.3
SD tissue viability (%)	9.8			0.1					2.8
CV (% viabilities)	9.8			3.2					2.9

* Blank corrected mean OD₅₇₀nm of the negative control corresponds to 100% absolute tissue viability

** Mean relative tissue viability of the three positive control tissues is≤20%

*** Standard deviation (SD) obtained from the 3 concurrently tested tissues is≤18%

Table 2: Historical data

	Mean OD570±30nm NK	Mean relative viability (%) PC	SD Viability (%)
Mean	1.843	4.3	4.2
SD	0.286	2.2	4.7
n	22	22	84

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study the test item, Harpin-ab Fermentation Extract, showed no skin irritant effects.