Propatylnitrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.12.2017 - 14.12.2017

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

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Protocol: EpiOcularTM EIT for the prediction of acute ocular irritation of chemicals

Version / remarks:

Version 9, June 29, 2015, MatTek corp.

Deviations:

no

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

Test animals / tissue source

Species:

human

Strain:

other: keratinocyte strain 4F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
Cell damage (cytotoxicity), playing an important, if not the primary, mechanistic role in determining the overall serious eye damage/eye irritation response of a chemical regardless of the physicochemical processes underlying tissue damage, is followed in this test.

This test uses an in vitro procedure allowing the identification of chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. This test is not able to distiguih between serious eye damage and eye irritation.


- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live

The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the 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 reconstructed human cornea-like epithelial model EpiOcular™ comes from MatTek, Bratislava, SK, supplied with Certificate of Analysis. Lot No. of tissues used for this test: 27017 kit B.

On the day of receipt, EpiOcularTM tissues were conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 62 minutes at standard culture conditions and, after media replacement, overnight (following 18 hours 28 minutes) also standard at culture conditions.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance (50 mg of substance/surface ratio 39.7 uL/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS. The test substance was spread over entire tissue surface.
A single testing, composed of two replicate tissues, was run.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25±2 mins immersion incubation (post-soak)
18 hours at standard culture conditions (post-treatment incubation)

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
Direct MTT reduction - functional check in tubes => The test was performed as a part of another study: Study No. 314/17/4AC: Propatyl nitrate - In vitro Skin Corrosion (EpiDermTM Model), VUOS-CETA Report No. 17-664, 2017. No direct-reducing properties were observed.
Colour interference
The test was performed as a part of another study: Study No. 314/17/4AC: 5-aminotetrazole - In vitro Skin Corrosion (EpiDermTM Model), VUOS-CETA Report No. 17-664, 2017. No change of colour was observed.
MTT test
A single testing, composed of 2 replicate tissues, is run (plus 3 for the positive control (PC) and 3 for negative control (NC)).

- RhCE tissue construct used, including batch number
The reconstructed human cornea-like epithelial model EpiOcular™ comes from MatTek, Bratislava, SK.
Lot No. of tissues used for this test: 27017 kit B

- Doses of test chemical and control substances used
The test substance (50 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS.
PC: Methyl Acetate 99%, MatTek, Lot No. 032817ISA, exp. 28/03/2018
NC: water for injection Ardeapharma, Lot. No. 1608120439 exp.08/2018

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2).
25 ± 2 minutes immersion incubation (post-soak) at room temperature
18 hours at standard culture conditions (37±1°C, 5±1 % CO2, humidified incubator)

- Description of any modifications to the test procedure:
Assay acceptance criterion was not fulfilled for absorbancies of extracts from positive control tissues. As all the tissues had viabilities under 50% of negative control viability we consider that this deviation has not impact on study results.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
OD570 is measured on a plate reader Biotek Epoch. Isopropyl alcohol serves as a blank. No external filter is used.

- Description of the method used to quantify MTT formazan
Optical density (OD570) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.
Then the mean relative tissue viability of two individual tissues exposed to the test substance is calculated – this value is, after correction, used for the comparison with limit value.
Tests for colour interference and direct reduction did not demonstrate influence of colour or reductive properties of the test item on study results. Thus, no steps for correction of results were performed.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Results should thus be interpreted as follows:
- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
- The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Positive and negative control means and acceptance ranges based on historical data
1) The negative control OD > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of control viability
3) The difference of viability between the two relating tissues of a single chemical is < 20% in the same run. This applies also to the killed controls and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Irritation parameter:

other: average viablility

Run / experiment:

1

Value:

101

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: i.e. viability was > 60 %

Other effects / acceptance of results:

The average negative control (neat extract) OD570 was 1.638 what is > 0.8 and < 2.5. This criterion was fulfilled.
The mean relative viability of the positive control was 20.0 % what is below 50% of negative control viability. This criterion was fulfilled.
The difference of viability between the three relating tissues of the negative control was 11.7 %. The difference of viability between the two relating tissues of the test substance was 12.4 % what is < 20%. These criteria were fulfilled. The difference of viability between the three positive control tissues was 21.0 % what is > 18%. This criterion was not fulfilled (for comment see Any other information ...above).

No problems occurred at treatment but part of the test substance remained not wetted after the treatment period. Bottom layer directly adjoin to tissue was wetted.
After rinsig, part of the test substance remained on tissue until post soak. During this step it was taken out.

Any other information on results incl. tables

Table 1: OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

The data presented are corrected by subtraction of OD570 isopropyl alcohol itself (blank, 0.040).

Code	Treatment	OD570			mean	SD	Viability %
		Tissue 1	Tissue 2	Tissue 3		%SD
NC	water	1.488	1.516	1.909	1.638	0.175	100.0
	% NC	90.9	92.5	116.6	100.0	11.7
C1	314/17	1.567	1.741		1.654	0.087	101.0
	% NC	95.7	106.3		101.0	5.3
PC	99% MA	0.321	0.247	0.414	0.327	0.069	20.0
	% NC	19.6	15.1	25.3	20.0	21.0

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test substance Propatyl nitrate was 101.0 % of negative control average value.
The effect of the test substance was negative in EpiOcularTM model (tissues were not damaged).
According to the classification criteria, the test substance, Propatyl nitrate, is identified as not requiring classification and labelling according to UN GHS (No Category). In this case no further testing in other test methods is required.

Executive summary:

The test substance, Propatyl nitrate, was assayed for the in vitro eye irritation in human cornea-like model EpiOcular^TM. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular^TMEye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).

After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the tissue and it was spread on the entire tissue surface. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2). Two tissues were used for the test substance and three for every control.

After removal of the test substance, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. Three hours incubation with MTT and 2-3 hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Tests for colour interference and direct reduction were performed as a part of another study (Study No. 314/17/4AC: Propatyl nitrate - In vitro Skin Corrosion (EpiDermTM Model), VUOS-CETA Report No. 17-664, 2017). Neither direct reducting properties nor colour interference with endpoint were found.

Under the above-described experimental design average viability of treated tissues was 101.0% i.e. viability was >60 %.

The effect of the test substance was negative in EpiOcular^TMmodel (tissues were not damaged).

According to the classification criteria, the test substance, Propatyl nitrate, is identified as not requiring classification and labelling according to UN GHS (No Category). In this case no further testing in other test methods is required.