Adenine

Endpoint summary

Description of key information

Skin irritation / corrosion

In a study according OECD TG 439, the test item is not identified as requiring classification and labelling according to UN GHS, as the mean percentage tissue viability was higher than 50% of the negative control (reference 7.3.1-1).

Eye irritation

In a study according OECD TG 492 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 (reference 7.3.2-1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

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 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

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

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.

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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)

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.

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.

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

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%

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

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).

Eye irritation

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.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data for skin irritation/corrosion and eye irritation/corrosion are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is neither considered to be classified for skin irritation/corrosion nor for eye irritation/corrosion (UN GHS: no category) under Regulation (EC) No 1272/2008, as amended for the eleventh time in Regulation (EU) No 2018/669.