CDX-403

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 July 2021 - 23 July 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes

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)

Test animals / tissue source

Species:

human

Strain:

other: normal human keratinocytes

Details on test animals or tissues and environmental conditions:

- Justification of the test method (e.g. ICE, EIT, RhCE) and considerations regarding applicability:
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 eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 31797 kit W + kit Y)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 54.5 to 65.2 mg

Duration of treatment / exposure:

6 hours ± 15 minutes at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:
Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed. The test item has been tested previously for possible direct MTT reduction and color interference in the Skin irritation using EpiSkinTM as a skin model (Skin irritation Test Facility Study No.20288972).
The solutions were turned blue / purple or a blue / purple precipitate was observed in presence of MTT, therefore it was concluded that the test item did interfere with the MTT endpoint.
The Optical Density (OD) for the test item solution was ≤0.08, therefore it was concluded that the test item did not interact with the MTT measurement.

Test System Set Up:
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room
temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium, Assay Medium was supplied by MatTek Corporation, Ashland, USA.

Application/Treatment of the Test Item:
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
In addition, since the test item reacted with the MTT medium, two freeze-killed tissues were
treated with test item and one freeze-killed non treated tissue were used per exposure time for the cytotoxicity evaluation with MTT.
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for minimal 30 minutes.
Two tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues with 50 μL Methyl Acetate (positive control) respectively.
At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure
period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly
rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minutes immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

Cell Viability Measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in
duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Calculation of Cell Viability:
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls
(ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

MTT Interacting Test Items:
Nonspecific MTT reduction (NSMTT) was calculated. NSMTT is the difference between the mean OD of the untreated freeze-killed tissues (ODkt_u+MTT) and test item treated freeze-killed tissues (ODkt_t+MTT) expressed as percentage of the mean of the negative control tissues (ODlt_u+MTT).
%NSMTT = [(ODkt_t+MTT – ODkt_u+MTT)/ mean ODlt_u+MTT] * 100
True tissue viability is calculated as the difference between the living test item treated tissues
incubated with MTT medium (ODlt_t+MTT) and the difference between ODkt_t+MTT and
ODkt_u+MTT.
OD= ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)
%Viability = [OD/ mean ODlt_u+MTT] * 100
In case the %NSMTT ≤ 0.0, there is no need to correct for interference of the test item.

ACCEPTABILITY CRITERIA
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be
> 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The difference between the % tissue viabilities of the two identically treated replicates should be <20.
d) The non-specific MTT reduction should be  50% relative to the negative control OD.

Interpretation:
The test chemical is identified as not requiring classification and labelling according to Regulation
(EC) No. 1272/2008 and its amendments if the mean percent tissue viability after exposure and p
ost-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
The test chemical is identified as “no prediction can be made” if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.

Results and discussion

In vitro

Results

Irritation parameter:

percent tissue viability 

Remarks:

mean

Run / experiment:

experiment 1

Value:

91

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

32%

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:
- Visible damage on test system: no

Interference of the Test Item with the MTT Endpoint:
Because the solutions did turn blue / purple and/or a blue / purple precipitate was observed and the OD for the test item solution was ≤0.08, therefore it was concluded that the test item did interfere with the MTT endpoint.
Since the test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), in addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 0.8% of the negative control tissues. The ODs of the test item treated viable tissues was corrected using the OD of the freeze-killed tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 32%.
The difference between the percentage of viability of two tissues treated identically was less than 10% for the test item. For the negative and positive control the difference between the percentage of viability of two tissues treated identically was 23 and 26% respectively, which is above the acceptability criteria of 20%. Since the individual viabilities of the tissues treated with the test item were clearly above 60%, this deviation did not influence the outcome of the test.

Any other information on results incl. tables

Mean Tissue Viability in the EpiOcular™ Test with CDX-403

	Mean tissue viability (percentage of control)	Difference between two tissues (percentage)
Negative control	100	23
Test item(1)	91	9.3
Positive control	32	26

(1) The test item values are corrected for the non-specific MTT reaction.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the outcome of an EpiOcular™ test performed according to OECD 492 guideline and GLP
principles, it is concluded that the substance is not irritating.

Executive summary:

An EpiOcular™ test was performed with the substance according to OECD guideline 492 and GLP principles. The substance was tested as it is. The mean cell viability of the positive control was 32%, and the mean cell viability of the negative control 100%. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the substance compared to the negative control tissues was 91%. Since the mean relative tissue viability for the substance was above 60% after 6 hours ± 15 minutes treatment, the substance is considered to be non-irritant and does not need to be classified according to Regulation (EC) No. 1272/2008 and its amendments.