Adenine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018 -08-01 to 2018-08-03

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

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic™ RHE-model RHE/S/17 (Batch No: 18-RHE-089), obtained from Episkin/SkinEthic Laboratories, Lyon, France.

Source strain:

other: not applicable

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 18-RHE-089
- Expiration date: 2018 -08-06
- Date of initiation of testing: 2018-08-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item, negative and positive control were removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper. The inserts were placed in 6-well plates with 2 mL fresh pre-warmed (room temperature) growth medium.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes)
- Spectrophotometer: microplate reader
- Wavelength: 570 nm
- Filter: filter test plate


NUMBER OF REPLICATE TISSUES: The test item as well as the positive and negative control were tested in batch-triplicates. Therefore, a total number of nine tissues was used in this study.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- No. of replicates : 3


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%.
A 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-treatment incubation is < 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

in vitro results: UN GHS:
Mean tissue viability <50% Category 2 or Category 1
Mean tissue viability >50% No Category

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg ± 2 mg per tissue

NEGATIVE CONTROL : Dulbecco's Phosphate-Buffered Saline
- Amount applied: 16 µL ± 0.5 µL per tissue
DPBS-buffer was used as negative control and as rinsing solution after treatment.

POSITIVE CONTROL : Sodium dodecyl sulfate
- Amount applied: 16 µL ± 0.5 µL per tissue
- Concentration: 5% aqueous solution of sodium dodecyl sulfate in deionised water

Duration of treatment / exposure:

42 minutes (± 1 minute)

Duration of post-treatment incubation (if applicable):

None

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

99.7

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
- Direct-MTT reduction: The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1: Results

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.612	102.5%	1.631	103.7%	1.475	93.8%	1.573	100.00	5.4%
Positive Control	0.022	1.4%	0.022	1.4%	0.022	1.4%	0.022	1.4%	0.0%
Test Item	1.510	96.0%	1.582	100.6%	1.614	102.6%	1.569	99.7%	3.4%

Table 2: Acceptability of the Test

	Acceptance Criterion	Result
Negative control OD	> 0.8and< 3.0	1.475to1.631

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

	Acceptance Criterion	Result
Mean OD negative control	> 1.2	1.573
Mean viability positive control	< 40%	1.4%
SD of group-mean value	< 18%	0.0% (positive control) 5.4% (negative control)

Acceptability of the Positive and Negative Control basedon Historical Data of the Testing Laboratory:

	Acceptance Criterion	Result
Mean OD negative control	> 1.444	1.573
Mean viability positive control	< 2.85%	1.4%

Test Item Data Acceptance Criteria:

	Acceptance Criterion	Result
SD of group-mean value	< 18%	3.4%

The study met all acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-compliant in vitro Skin Irritation: Reconstructed Human Epidermis Test according to OECD Guideline 439 and EU Method B.46 the test item was determined to be non-irritating to the skin.

Executive summary:

The skin irritating potential of the test item was assessed in a GLP-compliant in vitro Skin Irritation: Reconstructed Human Epidermis Test according to OECD Guideline 439 and EU Method B.46. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE-model were treated with the test item, a negative or a positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. A pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e.the test item is not a direct MTT reducer and the test item has no colorant properties. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the mean tissue viability was 99.7% (SD = 3.4%) and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: no category).