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

Skin irritation / corrosion

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 2018-01-04

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 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

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.

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 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

Results and discussion

In vitro

Results

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".

Any other information on results incl. tables

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

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 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.