Propatylnitrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13.09. – 04.10.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Adopted: July 28th, 2015

Deviations:

yes

Remarks:

Colour interference test was performed, which was not described in the Study Plan. This deviation had no impact on the outcome of study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Propatylnitrate

EC Number:

220-866-5

EC Name:

Propatylnitrate

Cas Number:

2921-92-8

Molecular formula:

C6H11N3O9

IUPAC Name:

2,2-bis[(nitrooxy)methyl]butyl nitrate (non-preferred name)

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Not available

IUPAC Name:

Unknown impurities

Test material form:

solid: particulate/powder

Details on test material:

white powder

In vitro test system

Test system:

human skin model

Remarks:

a reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek)

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Cell source:

other: Keranocyte strain: 00267

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SR)
- Tissue batch number(s): Lot No. 25841, kit B

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
tissues were thoroughly rinsed and blotted to remove the test substance/controls
Detailed procedure is described in internal SOP M/46/3.
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
tissues were transferred to 24-well plates containing MTT medium (1 mg·mL-1)
- Incubation time: 3 hr
- Spectrophotometer: Libra S22. Isopropyl alcohol serves as a blank.
- Wavelength: 570±30 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1. Direct MTT reduction - functional check in tubes: the test substance does not reduce MTT directly.
2. Colour interference: colour of the test substance did not interfere with evaluation
3. MTT test

DECISION CRITERIA
According to the OECD TG 431 as well as to the EU Method B.40, the test substance is considered to be corrosive to skin:
i) if the viability after 3 minutes exposure is less than 50 %, or
ii) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
The test substance is considered to be non-corrosive to skin:
i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

test substance C1: 25 mg of the test substance was placed directly atop to the previously moistened tissue (25 μL of H2O) and it was spread to match size of the tissue.
NC: 50 μL H2O tested with every exposure time
PC: 50 μL 8N KOH tested with every exposure time

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

180 minutes (in MTT medium)

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

86

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct MTT reduction - functional check in tubes:
The test substance did not change colour from red to blue, so other steps were not employed.

- Colour interference
The test substance did not change colour, so other steps were not employed.

ACCEPTANCE OF RESULTS: All assay acceptance criteria have been met.
Negative control: The assay meets the acceptance criterion - OD570 of the NC tissues was 1.746 (3 min) and 1.814 (60 min) which is ≥ 0.8 and ≤ 2.8.
Positive control: Viability of tissues treated with 8N KOH after 60 minutes treatment was 3.6 % which is ≤15%.
Coefficient of variance: CV values in all triplets of tissues were ≤ 0.3 (see Table 1).

Any other information on results incl. tables

MTT test:

OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%), coefficients of variance and relative viabilities

Treatment 3 min	OD570			mean	SD	CV	%NC
water (NC)	1.808	1.731	1.700	1.746	0.045	0.026	100.0
314/17 (C1)	1.801	1.774	1.703	1.752	0.035	0.020	100.3
8N KOH (PC)	0.079	0.075	0.068	0.074	0.005	0.061	4.2
Treatment 60 min	OD570			mean	SD	CV	%NC
water (NC)	1.788	1.764	1.891	1.814	0.055	0.030	100.0
314/17 (C1)	1.599	1.037	2.043	1.560	0.412	0.264	86.0
8N KOH (PC)	0.064	0.063	0.068	0.065	0.002	0.033	3.6

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, the test substance Propatyl nitrate was non-corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.

Executive summary:

The test substance Propatyl nitrate was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according to OECD Test Guideline 431, In Vitro Skin Corrosion: Human Skin Model Test, Adopted: July 28th 2015.

Interference of colour with the endpoint was performed in advance. The colour of the test substance did not interfere with the endpoint.

Direct reduction test in test tubes was performed simultaneously. Colour change of MTT medium was not observed. The test substance is not directly reducing.

In the MTT test, the test substance (25 mg) was placed atop the previously moistened tissue. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (C1), three for the positive control (PC) and three for the negative control (NC).

After rinsing, tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol for two hours at room temperature with shaking. OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, the average viability of tissues treated with the test substance Propatyl nitrate was 100.3 % of the negative control average value after 3 minutes treatment and 86.0 % after 60 minutes treatment.

The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.

In the experiment arrangement described above, the test substance Propatyl nitrate was non-corrosive in the EpiDermTM model.