Adenine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-08-09 to 2018-09-10

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

Test guideline 1

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:

09 October, 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Adenine

EC Number:

200-796-1

EC Name:

Adenine

Cas Number:

73-24-5

Molecular formula:

C5H5N5

IUPAC Name:

adenine

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. It was concluded that the EpiOcular™ EIT is able to correctly identify substances and mixtures not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.

Test system

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg per tissue

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 min post-soak and 18 hours (± 15 minutes) post-incubation

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number
: EpiOcular™ human cell construct (OCL-200, OCL-212) procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava - Slovakia, [phone], www.mattek.com.; Lot No.: 27062

- Doses of test chemical and control substances used
Test item: 50 mg
Neg. Control: 50 µL
Pos. Control: 50 µL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
Exposure: 6 hours at 37 ± 1 °C
Post-exposure immersion: 25 min at room temperature
Post-exposure incubation: 18 hours (± 15 minutes) at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Sterile deionised water (50 µL) was used as negative control concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan: 570 nm, 96-well plate reader

- Description of the method used to quantify MTT formazan: Optical density (OD) measurement of the MTT extracts.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
A test item is labeled as “non-irritant”, if the test item treated percent tissue viability determined by MTT assay is >60% relative to the negative control treated tissue viability. A test item is labeled as “irritant”, if the percent tissue viability determined by MTT assay is <60% relative to the negative control treated tissue viability.

- Complete supporting information for the specific RhCE tissue construct used

Lot No.: 27062
Tissue viability: OD (540-570)= 2.016 ± 0.011
Barrier function: ET-50= 30.71 min
Sterility: sterile

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Prior to routine use the technical proficiency of the test method was established by using proficiency chemicals according to OECD Test Guideline No. 492.

- Acceptance criteria:
The assay meets the acceptance criterion if the mean OD570 of the NC tissues is > 0.8 and < 2.5.
The assay meets the acceptance criterion if the mean relative viability of Positive Control tissues is <50% of negative control viability.
The assay meets the acceptance criterion if the difference of viability between the two relating tissues of a single chemical is < 20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Irritation parameter:

other: %viability

Run / experiment:

1st run

Value:

94.7

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:

OTHER EFFECTS:
- Visible damage on test system: No

Any other information on results incl. tables

Table: Results obtained after treatment of the reconstructed human cornea-like epithelium (RhCE) model with the test item along with the results for the negative and positive control

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
	OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	1.767	98.40%	1.823	101.60%	1.795	100.00%	2.26	3.20%
Positive Control	0.47	26.20%	0.573	31.90%	0.522	29.10%	4.03	5.70%
Test Item	1.787	99.60%	1.612	89.80%	1.7	94.70%	6.93	9.80%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results obtained and under the laboratory testing conditions using the EpiOcular™ model , the test item is not identified as requiring classification and labeling according to UN GHS as the mean percentage tissue viability is 94.7% and, thus, higher than 60% of the negative control.

Executive summary:

The in vitro eye irritation potential of the test item was evaluated using EpiOcular™ model (OCL-200-EIT) according to OECD TG 492. Tissues were incubated at standard culture conditions for 30 ± 2 minutes. Afterwards, 50 mg each of test item, 50 µL of negative control and positive control were dispensed directly atop EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 6 hours. All the treatments were maintained in duplicates. At the end of treatment time with test item, negative control and positive control, tissue inserts were dipped into a beaker with DPBS, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The cultures were then rinsed in the second and third beaker of DPBS. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium in a 12-well plate for 25 minutes immersion incubation (post-soak) at room temperature which was intended to remove any test item absorbed. At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted onto the absorbent material, and transferred to the appropriate well of a prelabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours (± 15 minutes) in CO2 incubator at 37 °C and 5% CO₂ (post-treatment incubation). After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO₂. The inserts were removed from the 24-well plate after 180 minutes (±10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 ml isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plate was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically. The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate. The results of the functional test and the printout of the measurement were included in the raw data of the study. Viability of tissues was calculated. The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. So no additional controls were maintained. Mean percentage viability of negative control, positive control and test item were 100 ± 2.26, 29.01 ± 4.03 and 94.7 ± 6.93 respectively. As the mean percentage viability of the test item was higher than 60% of the negative control it is not identified as requiring classification and labeling according to UN GHS. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. Based on the results obtained and under the laboratory testing conditions using the EpiOcular™ model, the test item is not identified as requiring classification and labeling according to UN GHS as the mean percentage tissue viability is higher than 60% of the negative control.