Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 12,2019 - July 9,2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate

EC Number:

603-290-2

Cas Number:

128625-52-5

Molecular formula:

C18H28N6OP2F6

IUPAC Name:

Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The SkinEthic™ RHE-model RHE/S/17 was obtained from Episkin/SkinEthic Laboratories, Lyon, France.
- Tissue batch number(s): 19-RHE-110

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 mL DPBS
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours (± 5 minutes)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: OD 1.3(CV= 1.7%)
- Barrier function: ET50=5.9h
- Morphology: Multi-layered, highly differentiated epidermis consisting of basal, spinous and granular layers, and a multi-layered stratum corneum.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%.
A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is < 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 16 mg ± 2 mg per tissue

NEGATIVE CONTROL
- Amount(s) applied: 16 µL ± 0.5 uL per tissue

POSITIVE CONTROL
- Amount(s) applied: 16 µL ± 0.5 uL per tissue

Duration of treatment / exposure:

42 minutes (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st experiment

Value:

64.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

1.3%

Other effects / acceptance of results:

- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1: Results

Group	Tissue 1		Tissue 2		Tissue 3		Mean		SD
	OD	viability	OD	viability	OD	viability	OD	viability	viability
Negative Control	1.845	1057%	1.753	100.5%	1.638	93.9%	1.745	100.0%	5.9%
Positive Control	0.023	1.3%	0.025	1.4%	0.021	1.2%	0.023	1.3%	7.7%
Test item	1.098	62.9%	1.087	62.3%	1.199	68.7%	1.128	64.6%	5.4%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin.

Executive summary:

A study was performed according to the OECD TG 439 to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, the tissue viability was 64.6% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).