5'-O-[bis(4-methoxyphenyl)benzyl]-2'-deoxy-N-(2-methyl-1-oxopropyl)guanosine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-23 to 2017-12-21

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28th July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

68892-41-1

Molecular formula:

C35H37N5O7

Test material form:

solid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Firstly, 25 µL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm²) of the test item was applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Remarks:

Dulbecco's phosphate buffered saline (DPBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (MatTek; EPI-200-SIT)
- Tissue batch number(s): 25850
- Date of initiation of testing: 2017-10-25

EpiDerm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 25849)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 101917TMB)
1x bottle of DPBS Rinse Solution (Lot No.: 062717MGKA)
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)



TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 min followed by room temperature under sterile flow until 60 ± 1 min incubation time was over
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Tissues were washed by filling and emptying the inserts 15 times with DPBS under a constant stream in about 1.5 cm distance from the tissue surface. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (final)
- Incubation time: 3 h ± 5 min
- Spectrophotometer: Plate spectrophotometer
- Wavelength: 570 nm with a filter band pass of maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Viable within acceptable test range (see Table 1 in box 'Any other information on materials & methods incl. tables')
- Barrier function: Acceptable (see Table 1 in box 'Any other information on materials & methods incl. tables')
- Contamination: None (see Table 1 in box 'Any other information on materials & methods incl. tables')

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after exposure and post-incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and post- incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

60 ± 1 minute

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

3 tissues per dose group

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

1.666

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:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (Acceptance criterion of mean absolute OD (570 nm): 0.8 ≤ OD ≤ 2.8)
- Acceptance criteria met for positive control: Yes (Acceptance criterion of relative tissue viability of 20% or less)
- Acceptance criteria met for variability between replicate measurements: Yes (Acceptance criterion of standard deviation for relative tissue viability of 18% or less)

For detailed results see Table 2 in box 'Any other information on results incl. tables'.

Any other information on results incl. tables

Table 2: Result of the Test Item N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.625	1.601	1.495	0.114	0.084	0.088	1.720	1.659	1.762
	1.644	1.592	1.528	0.113	0.084	0.088	1.721	1.634	1.762
OD570(Blank Corrected)	1.581	1.557	1.451	0.070	0.040	0.044	1.676	1.615	1.718
	1.600	1.548	1.484	0.069	0.040	0.044	1.677	1.590	1.718
Mean OD570of the Duplicates (Blank Corrected)	1.591	1.552	1.467	0.070	0.040	0.044	1.677	1.603	1.718
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.537*			0.051			1.666
SD OD570	0.063			0.016			0.058
Relative Tissue Viability [%]	103.5	101.0	95.5	4.5	2.6	2.9	109.1	104.3	111.8
Mean Relative Tissue Viability [%]	100.0			3.3**			108.4
SD Tissue Viability [%]***	4.1			1.0			3.8
CV [% Viability]	4.1			31.3			3.5

CV: coefficient of variation; OD: optical density; SD: standard deviation

^*                     Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^**         Mean relative tissue viability of the three positive control tissues is < 20%.

^***        Standard deviation obtained from the three concurrently tested tissues is ≤ 18%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine showed no irritant effects as the mean relative tissue viability (% negative control) was > 50% (108.4%) after 60-min treatment of the test item followed by a 42 h post-incubation period. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category” for skin irritation.

Executive summary:

In a primary dermal irritation study conducted according to guideline OECD 439, the reconstituted three-dimensional human epidermis EpiDerm™-Model (EPI-200-SIT) (was topically exposed to 25 mg of N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine (100.0% purity) for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained from three tissues compared to the corresponding negative control tissues concurrently treated with DPBS. The mean tissue viability after exposure and post-incubation periods to N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine was 108.4%, which, in accordance with UN GHS "No Category" for skin irritation, is considered as a non-irritant to the skin.