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Toxicological information

Skin irritation / corrosion

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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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 is20%

*** Standard deviation (SD) obtained from the 3 concurrently tested tissues is18%

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.