3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile

Eye irritation

Administrative data

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

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

Test material information

Constituent 1

Reference substance name:

3-({bis[bis(propan-2-yl)amino]phosphanyl}oxy)propanenitrile

EC Number:

600-337-9

Cas Number:

102691-36-1

Molecular formula:

C15H32N3OP

IUPAC Name:

3-({bis[bis(propan-2-yl)amino]phosphanyl}oxy)propanenitrile

Test material form:

liquid

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

- 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

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 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%

Results and discussion

In vitro

Results

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"

Any other information on results incl. tables

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

Applicant's summary and conclusion

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”.