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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-23 to 2018-05-14
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 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 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
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.

Test animals / tissue source

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.

Test system

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. As the results of the first experiment were borderline, the experiment was repeated.

- 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.: 27017 ,27027),
1x bottle EpiOcularTM assay medium (Lot No.: 121117ISA, 0301218ISA),
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 092817MGKA),

- Doses of test chemical and control substances used:
1. Negative Control: 50 µL Aqua dest.
2. Positive Control: 50 µL methyl acetate (CAS 79-20-9, Merck, 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%

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Relative Tissue Viability [%]
Run / experiment:
Mean of replicates
Value:
64.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: First experiment, borderline results (60 +/- 5%)
Irritation parameter:
other: Relative Tissue Viability [%]
Run / experiment:
Mean of triplicates
Value:
92.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Experiment II, 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% (64.7%). As the results were within the borderline values (60 ± 5%), a second experiment was performed to confirm the first results. The mean relative tissue viability (% negative control) of the second experiment was > 60% (92.7%). The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.661 (Experiment 1), 1.480 (Repeated Experiment)). The mean relative tissue viability (% negative control) of the positive control was < 50% (18.6% (Experiment 1), 22.3% (Repeated Experiment)). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (19.2%). For detailed information please refer to Table 1 and Table 2 in box "Any other information on results incl. tables".



Any other information on results incl. tables

Table 1: Main results first experiment

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570values

1.823

1.516

0.314

0.380

0.927

1.246

1.801

1.505

0.308

0.377

0.943

1.247

OD570values
(blank-corrected)

1.780

1.473

0.271

0.337

0.884

1.203

1.758

1.462

0.265

0.334

0.900

1.204

mean of the duplicates

1.769

1.468

0.268

0.336

0.892

1.203

mean OD

1.618*

0.302

1.048

TODTT

 -

 -

1.045

SD of mean OD

0.213

0.048

0.220

tissue viability [%]

109.3

90.7

16.6

20.7

55.1

74.4

relative tissue viability difference [%]***

18.6

4.2

19.2

mean tissue viability [%]

100.0

18.6**

64.7

mean tissue viability [%]
- NSClivingcorrected

 -

 -

64.5

Table 2: Main results second experiment

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570values

1.352

1.606

0.316

0.413

1.381

1.402

1.366

1.598

0.319

0.405

1.298

1.420

OD570values
(blank-corrected)

1.308

1.563

0.273

0.369

1.338

1.359

1.323

1.554

0.276

0.362

1.254

1.377

mean of the duplicates

1.769

1.468

0.274

0.365

1.296

1.368

mean OD

1.437*

0.320

1.332

TODTT

 -

 -

1.330

SD of mean OD

0.172

0.064

0.051

tissue viability [%]

91.5

108.5

19.1

25.4

90.2

95.2

relative tissue viability difference [%]***

16.9

6.3

5.0

mean tissue viability [%]

100.0

22.3**

92.7

mean tissue viability [%]
- NSClivingcorrected

 -

 -

92.5

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

* 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 (Experiment 1)

1.480

(Experiment 2)

0.8 < NK < 2.5 pass
Mean relative viability PC [%]

18.6

(Experiment 1)

22.3

(Experiment II)

< 50% pass
Max difference of % viability [%] 19.2 < 20% pass

Applicant's summary and conclusion

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 5′-O-[bis(4-methoxyphenyl)phenylmethyl]-2′-deoxythymidine, 3′-[2-cyanoethyl N,N-bis(1-methylethyl)phosphoramidite] (99.7% purity) 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-hour post-incubation period and compared to those of the concurrent negative controls. The mixture of 50 mg test item per 1 mL Aqua dest. showed colouring as compared to the solvent. Thus, coloured tissue controls were included and used for quantitative correction of results. The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (64.7%). As the results were within the borderline range of 60 +/- 5%, a second experiment was performed to confirm the results. The mean relative tissue viability (% negative control) of the second experiment was > 60% (92.7%). Based on the results, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.