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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-18 to 2018-01-08
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted on 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]
EC Number:
685-410-3
Cas Number:
98796-51-1
Molecular formula:
C40H49N4O8P
IUPAC Name:
5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N′-diisopropylphosphoramidite]
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 were 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)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 25849

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.: 101217ALB)
1x bottle of DPBS Rinse Solution (Lot No.: 092817MGKA)
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 the first 35 +/- 1 min, afterwards the plates were placed under the sterile flow until 60 +/- 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using 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
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue 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 DBPS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (Gibco, Cat. No. 14040-091, Lot No.: 1838067)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL SDS (MatTek, CAS No.: 151-21-3, Lot No.: 031617MGKA)
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 ± 1 min
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:
94
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:
For detailed results see Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Results of the Pre-Experiments:

The mixture of 25 mg of the test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT was determined to be 0%.

The mixture of 25 mg of the test item per 300 µL aqua dest. or per 300 µL isopropanol showed no colouring detectable by unaided eye assessment. Therefore, NSC was determined to be 0%.

Results of the main experiment:

Table 1: Result of the Test Item 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxythymidine, 3’-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite]

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.518

1.533

1.619

0.123

0.119

0.112

1.483

1.444

1.461

1.554

1.471

1.578

0.124

0.121

0.111

1.480

1.427

1.437

OD570(Blank Corrected)

1.475

1.489

1.576

0.080

0.075

0.068

1.439

1.400

1.418

1.510

1.428

1.535

0.080

0.078

0.067

1.437

1.383

1.394

Mean OD570of the Duplicates (Blank Corrected)

1.493

1.459

1.555

0.080

0.076

0.068

1.438

1.392

1.406

Total Mean OD570of 3 Replicate Tissues (Blank Corrected)

1.502*

0.075

1.412

SD OD570

0.049

0.006

0.024

Relative Tissue Viability [%]

99.4

97.1

103.5

5.3

5.1

4.5

95.7

92.6

93.6

Mean Relative Tissue Viability [%]

100.0

5.0**

94.0

SD Tissue Viability [%]***

3.3

0.4

1.6

CV [% Viabilities]

3.3

8.4

1.7

* Blank-corrected mean OD570 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 (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Table 2:  Quality Criteria

 

Value

Cut off

pass/fail

Mean Absolute OD570 nmNK

1.545

0.8 ≤ NK ≤ 2.8

pass

Relative Viability [%] PC

5.0

≤ 20%

pass

SD Viability[%]

0.4 -3.3

≤ 18%

pass

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions, 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxythymidine, 3’-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxythymidine, 3’-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] (purity 99.7%) for 60 mins followed by a 42 h post-incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was > 50% (94.0%). Based on this result, the test item is classified as a non-irritant according to the UN GHS.