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Description of key information

Skin irritation:

In a reliable GLP OECD Guideline 439 in vitro skin irritation study (Human Skin Model Test (EpiDerm™)), no evidence of skin irritant effects were observed. Therefore, the test item, Harpin-ab Fermentation Extract, was considered to be non-irritating to skin.

 

Eye irritation:

In a reliable GLP OECD guideline 437 in vitro eye irritation study (Bovine Corneal Opacity and Permeability Assay), the mean in vitro irritation score was -0.46,. Therefore, Harpin-ab Fermentation Extract is considered to be non-irritating to eyes.

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:
09 February - 13 March 2018
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
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.
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

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

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 March - 18 April 2018
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: lot# R134-2
- Expiration date of the batch: 17 July 2018
- Appearance: Tan, light brown liquid
- Active components: Harpin-ab proteins 0.4% in aqueous solution
- Storage condition of test material: ≤ -70°C, protected from light
- Treatment of test material prior to testing: The test item was thawed before application.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: Not specified
- Characteristics of donor animals: Not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Fresh eyes were collected from the slaughterhouse on the test day and were transported in HBSS (Hanks' balanced salt solution) containing penicillin/ streptomycin on ice to the laboratories.
- Time interval prior to initiating testing: Eyes were collected and the corneas prepared on the test day.
- Indication of any existing defects or lesions in ocular tissue samples: Any defective eyes and corneas were discarded.
- Indication of any antibiotics used: The HBSS contained penicillin and streptomycin
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL of the test substance
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
The corneas were incubated for 2 hours prior to the illuminance measurement and for a further 90 minutes prior to measurement of optical density.
Number of animals or in vitro replicates:
3 corneas per treatment group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. The corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS (fetal bovine serum) and 2 mM L-glutamine (complete RPMI). The prepared corneas were incubated for one hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS :
Before the corneas were mounted in corneal holders, they were visually examined for defects and any defective cornea were discarded. Only corneas that had an initial luminance reading l >l0/ 1.1651 lux were used for the assay.

NUMBER OF REPLICATES :
Three corneas were used for each treatment group (test substance and positive and negative controls)

NEGATIVE CONTROL USED:
Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative control corneas. The negative controls were treated with physiological saline 0.9% NaCl.

POSITIVE CONTROL USED
The positive controls were treated with ethanol 100%.

APPLICATION DOSE AND EXPOSURE TIME :
750 µL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1°C either the test substance or the control substance was removed.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE :
- Number of washing steps after exposure period: After the test substance or control substance was removed, the epithelium was washed at least three times with MEM (minimum essential medium, containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

POST-EXPOSURE INCUBATION:
Following rinsing of the cornea, an illuminance measurement was performed after 2 hours incubation at 32 ± 1°C. The medium was then removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI and the anterior chamber filled with 1 mL of a 4 mg/mL sodium fluorescein solution. The corneas were then incubated for a further 90 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measurement of illuminance
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry at 490 nm (OD490) .
- Others: Each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG were used.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of three measurements
Value:
-0.46
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:
EFFECTS:
None of the corneas treated with the test substance showed opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean.
- Range of historical mean in vitro irritation score values for positive control: Mean value (MV) - 2xSD = 29.20; MV + 2xSD = 67.94

Table 1: In vitro irritation score

Cornea No.

Test item

Corrected opacity

Corrected OD490 value

IVIS

1

Negative control

0.50

0.022

1.18

2

0.75

0.045

3

1.05

0.015

MV

0.77

0.027

4

Positive control

31.03

0.958

42.99

5

26.87

1.139

6

26.45

0.878

MV

28.12

0.991

7

Test item

-0.04

-0.007

-0.46

8

-0.33

-0.008

9

-0.52

-0.016

MV

-0.30

-0.011

MV = mean value

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the mean in vitro irritation score of -0.46, the test substance, Harpin-ab Fermentation Extract is not considered to be an eye irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The in vitro skin and eye irritation studies performed in accordance with OECD Guidelines 439 and 437, respectively, show that Harpin-ab Fermentation Extract is not irritating. Therefore, in accordance with Regulation (EC) No. 1272/2008, Harpin-ab Fermentation Extract does not require classification as a skin or eye irritant.

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