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EC number: 600-337-9 | CAS number: 102691-36-1
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- Toxicological Summary
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- Irritation / corrosion
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Endpoint summary
Administrative data
Description of key information
The potential of 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (target substance) 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 can be considered non-irritant to the skin and eye.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-10-18 to 2018-01-04
- 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 28 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
- Treatment of test material prior to testing: All EpiDerm tissues were gently blotted to remove moisture. The test item was applied undiluted. - 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 viability after exposure and 42 h post-treatment 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 42 h post-treatment 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): 30 µL
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30µl DPBS
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- 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
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean of three tissues
- Value:
- 92.8
- 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".
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study under the given conditions, 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite 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 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (Purity 99.6 %) for 60 mins and 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% (92.8%). Based on this result, 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite is classified as a non-irritant according to the UN GHS.
Reference
Results of the Pre-Experiments:
The mixture of 30 μL 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 30 μL 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%.
Table 1: Result of the Test Item 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite
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 |
1.546 |
1.379 |
1.411 |
1.554 |
1.471 |
1.578 |
0.124 |
0.121 |
0.111 |
1.541 |
1.349 |
1.396 |
|
OD570(Blank Corrected) |
1.475 |
1.489 |
1.576 |
0.080 |
0.075 |
0.068 |
1.503 |
1.336 |
1.367 |
1.510 |
1.428 |
1.535 |
0.080 |
0.078 |
0.067 |
1.498 |
1.306 |
1.353 |
|
Mean OD570of the Duplicates (Blank Corrected) |
1.493 |
1.459 |
1.555 |
0.080 |
0.076 |
0.068 |
1.500 |
1.321 |
1.360 |
Total Mean OD570of 3 Replicate Tissues (Blank Corrected) |
1.502* |
0.075 |
1.394 |
||||||
SD OD570 |
0.049 |
0.006 |
0.094 |
||||||
Relative Tissue Viability [%] |
99.4 |
97.1 |
103.5 |
5.3 |
5.1 |
4.5 |
99.9 |
87.9 |
90.5 |
Mean Relative Tissue Viability [%] |
100.0 |
5.0** |
92.8 |
||||||
SD Tissue Viability [%]*** |
3.3 |
0.4 |
6.3 |
||||||
CV [% Viabilities] |
3.3 |
8.4 |
6.8 |
* 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 NK | 1.545 | 0.8 ≤ NK ≤ 2.8 | pass | |
Relative viability PC [%] | 5.0 | ≤ 20% | pass | |
SD Viability [%] | 0.4 - 6.3 | ≤ 18% | pass |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-11-29 to 2018-01-29
- 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 28 July 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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
- Description of the test system used: The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium that is 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-keratinised surface, showing a cornea-like structure analogous to that found in vivo - 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 µL (undiluted) - 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. The EpiOcular™ tissues were then transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h at 37 ± 1 °C, 5.0% CO2 / 95% air. Then the tissues 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 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. Then the 6-well plate(s) were incubated for 30 ± 2 min at 37 ± 1 °C, 5.0% CO2 / 95% air. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS, occurring also in sequential order, e.g. in one-minute intervals. 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 12 ± 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 120 ± 15 min 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 pre-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, with 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 pierced and the liquid within each insert was decanted into the well from which it was taken.
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.: 27017)
1x bottle EpiOcularTM assay medium (Lot No.: 12111715A)
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 No. 79-20-9 (positive control; Lot No.: S694311)
3. Test Item 50 µL (undiluted)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 6 h at 37 ± 1 °C, 5.0% CO2 / 95% air.
Post exposure post-soak plate: 30 ± 2 min at 37 ± 1 °C
Post exposure post-treatment plate: 120 ± 15 min 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 two replicates
- Value:
- 108.4
- 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% (108.4%). 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" - Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study under the given conditions, 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite 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 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (99.6 % 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 µL 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 120 min post-incubation period and compared to those of the concurrent negative controls. The test item showed no reduction of MTT and no colouring potential. The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (108.4 %). Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.
Reference
Table 1: Main results | |||||||
Name | Negative control | Positive control | Test item | ||||
Tissue | 1 | 2 | 1 | 2 | 1 | 2 | |
OD570 values (blank-corrected) |
1.720 | 1.959 | 0.040 | 0.035 | 2.163 | 1.917 | |
1.808 | 1.952 | 0.040 | 0.032 | 2.121 | 1.865 | ||
mean of the duplicates | 1.764 | 1.956 | 0.040 | 0.034 | 2.142 | 1.891 | |
mean OD | 1.860* | 0.037 | 2.016 | ||||
SD of mean OD | 0.12 | 0.00 | 0.15 | ||||
tissue viability [%] | 94.9 | 105.1 | 2.1 | 1.8 | 115.2 | 101.6 | |
relative tissue viability difference [%]*** | 10.3 | 0.3 | 13.5 | ||||
mean tissue viability [%] | 100.0 | 2.0** | 108.4 |
* 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: Quality Criteria | ||||
Value | Cut-off | pass/fail | ||
Mean absolute OD570 NK | 1.904 | 0.8 < NK < 2.5 | pass | |
Relative viability PC [%] | 2.0 | < 50% | pass | |
SD Viability [%] | 13.5 | < 20% | pass |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Additional information
The potential of 2-Cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (target substance) 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 can be 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).
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