CDX-403

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 Jun 2021 - 21 Jun 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

14 June 2021

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Halohydrin dehalogenase

Cas Number:

132421-41-1

IUPAC Name:

Halohydrin dehalogenase

Test material form:

solid: particulate/powder

Details on test material:

Identification: CDX-403
Physical Description: Light brown powder
Storage Conditions: In freezer (≤ -15°C)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Details on animal used as source of test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 21-EKIN-024 & 21-EKIN-022)

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is
recommended to minimize the need of in vivo testing. One of the validated in vitro skin
irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g.
OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Adult human donors
- Absence of HIV1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen HBs, bacteria, fungus and mycoplasma
- This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which will be seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Expiration date: 21June 2021 (21-EKIN-024) and 07 June 2021(21-EKIN-022)

KILLED TISSUES
Living epidermis (21-EKIN-022) was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ±1 hours. After incubation, killed epidermis was stored in a freezer set to maintain -20°C.
Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates
on 2 mL maintenance medium. Further use of killed tissues was similar to living tissues.

TEMPERATURE USED FOR TEST SYSTEM
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, at 37.0 ± 1.0°C (actual range 36.1 - 39.7°C)
- Humid atmosphere of 80-100% (actual range 76 - 91%) containing 5.0 ± 0.5% CO2 in air in the dark

APPLICATION/TREATMENT OF THE TEST ITEM
The test was performed on a total of 3 tissues per test item together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water to ensure close contact of the test item to the tissue and the solid test item (11.2 to 19.5 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Since the test item reacted with the MTT medium, in addition three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.

TEST FOR COLOR INTERFERENCE BY THE TEST ITEM
- To assess the color interference, 50 mg of the test item or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water.
- The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark.
- 50 mg of the test item or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL
isopropanol.
- The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking.
-At the end of the exposure time, the mixtures were centrifuged for 30 seconds at 16000 g if
needed and the absorbance of the solutions was determined spectrophotometrically at 570 nm
in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

TEST FOR REDUCTION OF MTT BY THE TEST ITEM
- 50 mg of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline).
- The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark.
- A negative control, 50 μL sterile Milli-Q water was tested concurrently.
- If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 hours at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤1.5) and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
d) The non-specific MTT reduction should be  30% relative to the negative control OD.

INTERPRETATION (See Table 1)

A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

11.2 to 19.5 mg applied directly on top of the skin tissue. The test item was spread to match the size of the tissue

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 1 hours at 37°C

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 replicates

Value:

80

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

19

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

See "attached full study report" for detailed results and historical control data.

- OTHER EFFECTS:
- Direct-MTT reduction:
A color change was observed in the presence of MTT therefore it was concluded that the test item interacted with the MTT endpoint. Three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 17% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

- Colour interference with MTT:
The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0799 and 0.0040, respectively. Therefore it was concluded that the test item did not induce color interference.


ACCEPTABILITY CRITERIA
The positive control had a mean cell viability of 19% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 11%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The substance does not cause skin irritation in the in vitro skin irritation test (OECD guideline 439).

Executive summary:

In an in vitro skin irritation test, performed according to OECD guideline 439 and in accordance with GLP principles, the ability of the substance to induce skin irritation was tested using reconstructed human epidermis (EPISKIN-SM). The test substance was applied to 0.38 cm² cultured skin. After 15 minutes exposure, the substance was removed and the tissues were incubated for 42 hours. Survival of unexposed tissue (negative control) was set at 100%. The positive control had a mean tissue viability of 19%. The test substance showed a mean tissue viability of 80% compared to the negative control. Since the mean relative tissue viability after exposure to the test substance was above 50%, no classification is required for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).