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

Endpoint summary

Description of key information

The potential of the target substance N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine (100% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 492) was tested in suitable in vitro test methods. Based on the results, the target substance is considered non-irritant to the skin and eye.

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:

2017-10-23 to 2017-12-21

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:

adopted 28th July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

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

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'.

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%.

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.

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:

2017-11-21 to 2018-01-11

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:

adopted 28th July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Approximately 50 mg (83.3 mg/cm²) of the test item were applied directly atop the EpiOcular™ tissue using an application spoon avoiding compression of the test item. The test item was spread to match the size of the tissue.

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method: This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

Duration of treatment / exposure:

6 ± 0.25 h

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

- Details of the test procedure used:
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air for 16 - 24 h. After the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye. Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. The test item was applied on the tissues placed on a sterile surface. Start time was recorded with dosing of the first tissue staggered in e.g. one-minute intervals. After dosing, the tissues were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 +/- 1 °C, 5.0% CO2 / 95% air until the 6 ± 0.25 h of the first dosed tissue was over. At the end of the exposure period the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 +/- 1°C, 5.0% CO2 / 95% air.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h +/- 10 min at 37 +/- 1 °C, 5.0% CO2 / 95% air. After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract the formazan only from the bottom of the tissues to avoid possible contamination of test material. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item. Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings. For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

- RhCE tissue construct used, including batch number:
EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek), consisting of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 27015)
1x bottle EpiOcularTM assay medium (Lot No.: 11271715A)
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 092817MGKA)
1x vial methyl acetate, CAS No. 79-20-9 (positive control; Lot No.: S6943111)

- Doses of test chemical and control substances used:
1. Negative Control 50 µL Aqua dest.
2. Positive Control 50 µL methyl acetate (CAS No. 79-20-9 (positive control; Lot No.: S6943111)
3. Test Item 50 mg

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air.
Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
See section "Pre-experiments" in box "Any other information on materials and methods incl. tables"

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable):
2 tissues per group

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer):
570 nm ± 30 nm

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability (% negative control) <= 60 %: Irritant (I): UN GHS “Category 1” or “Category 2”
Mean tissue viability (% negative control) > 60%: Non-Irritant (NI): UN GHS “No Category”

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
Historical control data were generated from 2012-2016:
Absolute OD570 ± 30 nm NK: Mean: 1.664; SD: 0.311, n= 14
Relative Viability PC [%]: Mean: 28.9, SD: 12.0, n= 14
Difference of Viability [%]: Mean: 9.9, SD: 15.9, n= 53

Test Acceptance Criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
- mean relative tissue viability of the positve control is < 50%
- relative tissue viability difference of replicate tissues is < 20%

Irritation parameter:

other: Relative tissue viability (%)

Run / experiment:

Mean of replicates

Value:

96.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (96.2 %). For detailed information please refer to Table 1 in box "Any other information on results incl. tables".

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
See also Table 2 in box "Any other information on results incl. tables"

Table 1: Main Results
Name	NK		PC		TM
Tissue	1	2	1	2	1	2
OD570values (blank-corrected)	1.818	1.737	0.312	0.365	1.774	1.590
	1.847	1.704	0.328	0.356	1.837	1.634
Mean of the duplicates	1.833	1.720	0.320	0.360	1.806	1.612
Mean OD	1.776^*		0.340		1.709
SD of mean OD	0.07		0.02		0.12
Tissue viability [%]	103.2	96.8	18.0	20.3	101.6	90.7
Relative tissue viability difference [%]^***	6.3		2.3		10.9
Mean tissue viability [%]	100.0		19.1^**		96.2

* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

** mean relative tissue viability of the positive control is < 50%

*** relative tissue viability difference of replicate tissues is < 20%

Table 2: Acceptance Criteria
		Value	Cut-off	pass/fail
Mean absolute OD570 NK		1.661	0.8 < NK < 2.5	pass
Mean relative viability PC [%]		18.6	< 50%	pass
Max difference of % viability [%]		18.6	< 20%	pass

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category” for eye irritation.

Executive summary:

In the present study the eye irritant potential of N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6 hours exposure and 18 hours post-incubation period and compared to those of the concurrent negative controls. The test item showed no non-specific reduction of MTT and no colouring potential. Therefore, NSMTT was determined to be 0%.The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (96.2 %). Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

The potential of the target substance N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine(100% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 492) was tested in suitable in vitro test methods. Based on the results, the target substance is considered non-irritant to the skin and eye.

Justification for classification or non-classification

Based on available data, no classification for skin and/or eye irritation is warranted based on the results from suitable in vitro tests (OECD 439, 492).