Adenosine phosphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 October - 5 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

28 July 2015

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.

Version / remarks:

10 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Adenosine phosphate

EC Number:

200-500-0

EC Name:

Adenosine phosphate

Cas Number:

61-19-8

Molecular formula:

C10H14N5O7P

IUPAC Name:

adenosine phosphate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

solid: crystalline

Remarks:

crystalline powder

Details on test material:

Batch No: 11679700
Storage: at room temperature, protected from light

Test animals / tissue source

Species:

other: The EpiOcular™ human cell construct (MatTek Corporation)

Details on test animals or tissues and environmental conditions:

DETAILS ON TEST SYSTEM:
- RhCE tissue construct used: EpiOcular™ (OCL-200-EIT)
- Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia
-Lot No.: 27007
- Expiry date: 05 October 2017
- Storage: The EpiOcular™ (OCL-200-EIT) units were stored at refrigerator (2-8°C) until the preparation of tissues for treatment is started. The assay media supplied with the kits were stored at refrigerator (2-8°C).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
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. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both 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. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6h (± 15 min)

Duration of post- treatment incubation (in vitro):

25 ± 2 minutes immersion incubation (Post-Soak)
18 h ± 15 minutes (Post-treatment Incubation)

Number of animals or in vitro replicates:

Two

Details on study design:

DETAILS ON THE TEST PROCEDURE USED:

- Preparation of EpiOcular™ Tissues for Treatment
After the test kit arrival, the tissues were equilibrated to room temperature for about 15 minutes. The Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37°C in an incubator with 5±1% CO2, 90±10% humidified atmosphere for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours).

- Application
Two replicates were used for the test item and control(s) respectively.
Pre-Treatment: After the overnight incubation, the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Test Item: Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. To avoid that test item is spilled into the medium under the tissue inserts, the tissue insert was removed from the medium, placed onto a sterile surface (e.g. the lid of a microtiter plate) and dosed by pouring the solid test article onto the tissue surface so that the surface of the tissue was completely covered by the test item. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.
Positive and Negative Control: A volume of 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the EpiOcular™ tissues surface if necessary.

- Exposure
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37°C in an incubator with 5 % CO2, 90±10% humidified atmosphere).

- Rinsing
After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as following way: Three clean beakers (glass or plastic with minimal 150 mL capacity), containing a minimum of 100 mL each of Ca++Mg++ Free-DPBS were used per test item. Each test item utilizes a different set of three beakers. The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps (taking care of not to damage the tissues by the forceps). The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues was dipped into the first beaker of DPBS and was swirled in a circular motion in the liquid for approximately 2 seconds and after was 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 tissue was rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).

-Post-Soak and Post-incubation
After rinsing, the tissues were 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 minute immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
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 on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37°C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- MTT Test After Post-incubation
After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 10 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, 90±10% humidified atmosphere.

- Formazan Extraction
Each insert was removed from the 24-well plate after 3 hours ± 10 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with parafilm (between the plate cover and upper edge of the wells)
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.

- Cell Viability Measurements
Following the formazan extraction, 200 µL sample(s) from each tube (2×200 µL) was placed into the wells of a 96-well plate (labelled appropriately) and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm (±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using isopropanol solutions as the blank (8×200 µL).

OTHER INFORMATION ON MATERIALS AND METHODS

- Check-method for possible direct MTT reduction by the test Item
Approximately 50 mg test item was added to 1 mL MTT 1 mg/mL solution in a 6 well plate or tube and the mixture was incubated in the dark at 37±1 °C, 5±1% CO2, 90±10% humidified atmosphere. The mixture was incubated for approximately three hours and then any colour change observed:
- Test item which do not interact with MTT: yellow
- Test item interacting with MTT: blue or purple
If the MTT solution’s colour becomes blue or purple, the test item interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed tissues).

- Check-method to detect the colouring potential of test Item
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. Blue, dark purple and black test articles may be directly tested on colorant controls without further tests because it is obvious that they can interfere with the blue MTT product.
Approximately 50 mg test item was added to 1mL of water in a tube and the mixture was incubated in the dark at 37±1 °C, 5±1% CO2, 90±10% humidified atmosphere for one hour and then colour checked (unaided eye assessment).
Approximately 50 mg test item was added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in tubes. The tube was placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature and then colour checked (unaided eye assessment).

- Assay acceptance criteria
- The mean OD value of the two negative control tissues should be between 0.8 and 2.5.
- The acceptable percentage viability for positive control (mean of two tissues) is:
30 minute exposure: below 50% of control viability (liquids)
6 hours exposure: below 50% of control viability (solids)
- 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: Mean Tissue Viability (% of negative control)

Value:

88

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Possible direct MTT reduction with test item:
No colour change was observed after three hours of incubation during the check for possible direct MTT reduction by the test item. The test item did not interact with MTT, therefore additional controls and data corrections were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test item:
The test item showed no ability to become coloured in contact with water. The isopropanol became slightly opalescent after the contact with test item. However, this change was not significant and the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD value 1344
- Acceptance criteria met for positive control: 38 % viability at 6 hours exposure
- Difference of viability between the two tissue replicates: 2.3% to 7.3%.

Any other information on results incl. tables

Cell Viability

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below (Please fill tables below):

Negative Control: Sterile deionized water

Replicate	Optical Density (OD)	Viability (%)	Delta %
1	1.393	104	7.3
2	1.295	96	7.3
Mean	1.344	100

Positive Control: Methyl acetate

Replicate	Optical Density (OD)	Viability (%)	Delta %
1	0.469	35	6.2
2	0.552	41	6.2
Mean	0.510	38

Test Item

Replicate	Optical Density (OD)	Viability (%)	Delta %
1	1.196	89	2.3
2	1.166	87	2.3
Mean	1.181	88
Standard deviation (SD)		1.62

Remarks: delta %: The difference of viability between the two relating tissues

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, Adenosine-5’-monophosphate (AMP) (CAS No.: 61-19-8) is, thus, considered as non-irritant to eye (UN GHS No Category).

Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test item Adenosine-5’-monophosphate (AMP) (CAS No.: 61-19-8) on three-dimensional RhCE tissue in the EpiOcular™ model in vitro.

Before treatment the tissues were pre-wetted with approximately 20 µL of Ca⁺⁺Mg⁺⁺Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37±1°C in an incubator with 5±1% CO₂, 90±10% humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca⁺⁺Mg⁺⁺Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1°C in an incubator with 5±1% CO₂protected from light, 90±10% humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item showed no ability to become coloured in contact with water and isopropanol. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (=) to 60% compared to the negative control. Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.

The test item Adenosine-5’-monophosphate (AMP) did not show significantly reduced cell viability in comparison to the negative control (mean viability: 88 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to eye.

Positive and negative controls showed the expected cell viability values within acceptable limits.The experiment was considered to be valid.

 

The results obtained from this in vitroeye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions According to the current OECD Guideline No. 492, Adenosine-5’-monophosphate (AMP) (CAS No.: 61-19-8)is, thus, considered as non-irritant to eye (UN GHS No Category).