p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 September - 04 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

26 June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EU) No 2019/1390 of 31 July 2019 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH),

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 131: EpiSkinTM Skin Irritation Test 15 min – 42 hours

Version / remarks:

09 June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

impurity 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

impurity 2

Reference substance name:

Acetone

EC Number:

200-662-2

EC Name:

Acetone

Cas Number:

67-64-1

Molecular formula:

C3H6O

IUPAC Name:

acetone

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Constituent 1

Reference substance name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

EC Number:

269-198-6

EC Name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Cas Number:

68189-42-4

Molecular formula:

C6H6NO6P.2C4H11NO3

IUPAC Name:

4-nitrophenyl dihydrogen phosphate - 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Test material form:

solid

Details on test material:

Batch / Lot No. 46587100

Specific details on test material used for the study:

Batch-No.: 46587100
Storage: Refrigerator

In vitro test system

Test system:

human skin model

Remarks:

EpiSkinTM Small Model (three-dimensional human epidermis model) manufactured by EPISKIN Laboratories Lyon, France

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: EPISKIN Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 20-EKIN-036
- Expiry date: 07 September 2020
- Date of initiation of testing: 02 September 2020

PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated over night at 37±1°C in an incubator with 5±1% CO2, >= 95% humidified atmosphere.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Three replicates were used for the test item and positive and negative controls, respectively.

REMOVAL OF TEST MATERIAL AND CONTROLS (day 0)
- Volume and number of washing steps: 25 mL PBS 1x solution, once. rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source.
- Observable damage in the tissue due to washing: none

POST-INCUBATION (day 0-2)
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37±1°C in an incubator with 5±1% CO2, >=95% humidified atmosphere.

MTT TEST (day 2)
After the 42 hours (±1h) incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37±1°C in an incubator with 5±1% CO2 protected from light, >=95% humidified atmosphere.

FORMAZAN EXTRACTION (day 2)
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for approximately four hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.

CELL VIABILITY MEASUREMENTS (day 2)
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm using acidified isopropanol solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA:
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Test Item: The epidermal surface was first moistened with 5 µL deionised water* in order to improve further contact between powder and epidermis. Subsequently, approximately 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
*prepared by MILLIPORE Synergy UV HF ASTM 1: F8JA80461C in Toxi-Coop ZRT.

POSITIVE CONTROL
- Amount: 10µL
- Concentration: SDS 5 % aq.

NEGATIVE CONTROL
- Amount: 10µL
- Concentration: 1X PBS

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42h (± 1h)

Number of replicates:

Three replicates were used for the test item and controls, respectively.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

93

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean viability value of three tissues

Other effects / acceptance of results:

OTHER EFFECTS
- Possible direct MTT reduction with test item: Yellow colour was observed after three hours of incubation. Therefore, the test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test item: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white to slightly yellowish and therefore considered not to be able to significantly stain the tissues and lead to a false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: Mean OD value 0.836 and standard deviation value (SD) for the % viability 10.28 (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be = or < 18)
- Acceptance criteria met for positive control: 0.150 % mean viability range standard deviation value (SD) for the % viability 1.15 (The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be = or < 18.)
- For test chemicals, the standard deviation value (SD) of the % viability should be = or < 18: 5.14 % SD

Any other information on results incl. tables

Cell viability The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

Negative Control (1X PBS)

Replicate	Optical Density	Viability (%)
1	0.747	89
2	0.919	110
3	0.843	101
mean	0.836	100
standard deviation (SD)		10.28

 

Positive control  (SDS 5% aq.)

Replicate	Optical Density	Viability (%)
1	0.159	19
2	0.152	18
3	0.140	17
mean	0.150	18
standard deviation (SD)		1.15

Test item

Replicate	Optical Density	Viability (%)
1	0.729	87
2	0.798	95
3	0.808	97
mean	0.778	93
standard deviation (SD)		5.14

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item 4-NPP, di-TRIS (CAS 68189-42-4) indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item 4-NPP, di-TRIS (CAS 68189-42-4) is considered to be non-irritant to skin and is therefore not classified (UN GHS / CLP No Category).

Executive summary:

EpiSkinTM SM test of 4-NPP, di-TRIS (CAS 68189-42-4) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 26 June 2020.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2, = 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1 °C in 5±1 % CO2, = 95% humidified atmosphere and protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1× PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50 % of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item 4-NPP, di-TRIS did not show significantly reduced cell viability in comparison to the negative control (mean value: 93 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model , with the test item 4-NPP, di-TRIS (CAS 68189-42-4) indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item 4-NPP, di-TRIS (CAS 68189-42-4) is considered to be non-irritant to skin and is therefore not classified (UN GHS / CLP No Category).