3,7-dihydropurin-6-one;sodium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-05 to 2018-01-04

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 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:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dihydropurin-6-one;sodium

Cas Number:

45738-97-4

Molecular formula:

C5H3N4NaO

IUPAC Name:

3,7-dihydropurin-6-one;sodium

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White powder
- Purity / Composition: 98%
- Test item storage: Room temperature

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method: The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified 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. 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: 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): 52.9 - 59.3 mg

Duration of treatment / exposure:

6 hours + / - 15 minutes

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 hours ± 15 minutes at 37 °C

Number of animals or in vitro replicates:

2 tissues

Details on study design:

Experimental Design:
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.

Test for Color Interference by the Test Item:
Sodium hypoxanthine was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, approximately 50 mg of Sodium hypoxanthine 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. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. Furthermore, approximately 50 mg of Sodium hypoxanthine 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. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Centrifugation was not considered necessary. 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:
Sodium hypoxanthine was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 50 mg of Sodium hypoxanthine 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. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

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, which was refreshed after approximately 1 hour. Assay Medium was supplied by MatTek Corporation, Ashland, USA.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 68 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 - 37.1 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation:
No correction was made for the purity/composition of the test item. The solid test item (52.9 to 59.3 mg) was applied directly on top of the skin tissue. Sodium hypoxanthine was spread to match the size of the tissue. Any residual volumes were discarded.

Application 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. 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 30 ± 2 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 Sodium hypoxanthine (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. 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.

Results and discussion

In vitro

Results

Irritation parameter:

other: Relative Tissue Viability (%)

Run / experiment:

Mean of replicates

Value:

1.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with Sodium hypoxanthine compared to the negative control tissues was 1.6%. Since the mean relative tissue viability for Sodium hypoxanthine was below 60% it is considered to be potentially irritant or corrosive to the eye.
The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 29%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly.

OTHER EFFECTS:
Sodium hypoxanthine was checked for possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that Sodium hypoxanthine did not interact with the MTT endpoint. Sodium hypoxanthine 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 0.0083 and 0.0004, respectively. Therefore it was concluded that the test item did not induce color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1: Mean Tissue viability in the EpiOcular™ Test

	Mean tissue viability (percentage of control)	Standard deviation  (percentage)
Negative control	100	9.2
Test item	1.6	0.08
Positive control	29	0.61

Applicant's summary and conclusion

Interpretation of results:

other: UN GHS Category 1 or Category 2

Conclusions:

In this study under the given conditions, the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.

Executive summary:

In the present study the eye irritant potential of Sodium hypoxanthine (98% purity) was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.The test item showed no non-specific reduction of MTT and no colouring potential. Therefore, no additional controls for correction of results were necessary. Sodium hypoxanthine showed irritant effects. The mean relative tissue viability of two replicates (% negative control) was not < 60% (1.6%). Therefore, the test item is considered to be irritating to the eye in accordance with UN GHS “Category 1" or "Category 2”.