Registration Dossier

Administrative data

Description of key information

The In Vitro Irritancy Score (IVIS) for Propatyl nitrate was 0.

This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.

Under the above-described experimental design, the average viability of tissues treated by the test substance, Propatyl nitrate, was 89.0 % of negative control average value i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model.According to the classification criteria given in this report, the test substance is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.

Based on in vitro / ex vivo tests the test substance _____________________________ was non-irritating for skin and eye.

navíc 413-17-4AC_fin_EpiDerm_In vitro Skin Corrosion

Validated and accepted in vitro tests for eye corrosion/irritation and in vitro skin irritation have been conducted.

It was In vitro Skin Irritation Test (OECD TG No. 439) and In vitro Eye Irritation Test (OECD TG No.492)

and BCOP test (OECD TG No. 437, EU B47??????).

- Study No. 413/17/5AI: 5 -aminotetrazole - In vitro Eye Irritation Test (EpiOcularTM Model); VUOS-CETA Report No. 18 -12, 2018. Result: substance potentially requiring classification and labelling, further testing with other test methods required Under the above-described experimental design average viability of treated tissues by the test substance 5-aminotetrazole was 2.3 % of negative control average value i.e. viability was ≤ 60 %. The effect of the test substance was positive in EpiOcularTM model (tissues were damaged).According to the classification criteria, the test substance, 5-aminotetrazole, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.

- Study No. 413/17/4AI: 5-aminotetrazole - In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 17-780, 2017. Result: no category in regard to skin irritation

- Study No. 413/17/5BCOP: 5-aminotetrazole - Bovine Corneal Opacity and Permeability Test; VUOS-CETA Report No. 17-760, 2017

Result: no prediction can be made

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
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 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
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).

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

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.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26.09.2017 - 29.09.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted: 28th July, 2015
Deviations:
yes
Remarks:
Direct reduction in test tubes was not performed because it was performed as a part of another study – a deviation from Study Plan.
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No. 761/2009, 23rd July 2009
Qualifier:
according to guideline
Guideline:
other: MatTek Protocol for: INVITRO EpiDermTM SKIN IRRITATION TEST For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm, Model EPI-200-SIT
Version / remarks:
Rev. 26/3/2012,1-37
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: tissue for research puposes from accredited institutions
Source strain:
other: Keratinocyte strain 00267
Vehicle:
physiological saline
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Tissues: the reconstructed human epidermal model EpiDermTM (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 25844 kit A

TEMPERATURE USED FOR TEST SYSTEM
culture conditions 37±1°C, 5±1 % CO2, moistened tissue

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: thoroughly rinsed with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg·mL-1
- Incubation time: 180±5 mins
- Spectrophotometer: Libra S22 at 570 nm. Isopropyl alcohol serves as a blank.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Based on Certificate of Analysis the model passed all parametres for viability, barrier function, sterility.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
The test substance was fully rinsed off the tissues. No changes in tissue appearance were observed during the experiment.
The first tissue treated with the test substance was pierced already during treatment. The tissue was not damaged but it was partially separated from insert and partially floated in medium adhered on plastic bottom. Such tissues use to have lower OD570.
MTT test: a single testing, composed of three replicate tissues, was run.

PREDICTION MODEL / DECISION CRITERIA
OECD Test Guideline No. 439 (1), par. 36:
- In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2.
- The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 mg of the test substance was placed directly on previously moistened tissue with 25 µL of PBS/tissue and spread on the tissue surface.
NC: PBS (phosphate buffered saline), prepared in laboratory 29/09/17 exp. 29/03/18 (washing of tissues) and MatTek DPBS lot. No. 062717MGKA, exp. 27/06/2017 (wetting of tissues, negative control)
PC: 5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 092117ALF, exp. 05/10/2017
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
43 hours 23 mins
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
89
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All study acceptance criteria were fulfilled.
The mean OD570 of the NC tissue was 1.926 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.
The mean viability of the PC tissues expressed as % of the negative control tissues was 2.2 % which meets the acceptance criterion of ≤ 20 %.
The SD calculated from individual % tissue viabilities of the 3 identically treated replicates was 0.1 % for the positive control, 5.1 % for negative control and 11.9 % for the test substance (rather higher value caused by the non-standard tissue) what is < 18 % in all cases.

MTT test

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

Treatment

OD570

 

 

 Avg

SD

%NC

PBS (NC)

2.059

1.822

1.896

1.926

0.099

 

viability (%NC)

106.9

94.6

98.5

100.0

5.141

100.0

314/17 (C2)

1.391

1.850

1.899

1.713

0.229

 

viability (%NC)

72.2

96.1

98.6

88.97

11.882

89.0

5% SDS (PC)

0.039

0.043

0.044

0.042

0.002

 

viability (%NC)

2.0

2.2

2.3

2.18

0.112

2.2

PBS - phosphate buffered saline

SDS - sodium dodecyl sulphate

Interpretation of results:
GHS criteria not met
Conclusions:
Under the above-described experimental design, the average viability of tissues treated by the test substance, Propatyl nitrate, was 89.0 % of negative control average value i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model.
According to the classification criteria given in this report, the test substance is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.
Executive summary:

The test substance, Propatyl nitrate, was assayed for in vitro skin irritation in the human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test Test for use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

After pre-incubation of tissues, 25 mg of the test substance was placed directly on tissue and spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.

After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed. Optical density (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.

No complementary experiments for correcting of colour interference and direct reduction were done, because they were performed as a part of the Study No. 314/17/4AC: Propatyl nitrate - In vitro Skin Corrosion Test (EpiDermTM Model); VUOS CETA Report No.: 17-664. These experiments confirmed no direct reduction by the test substance and no color interference with the endpoint.

Under the above-described experimental design, average viability of treated tissues was 89.0 %, i.e. viability was > 50 %.

The effect of the test substance was negative in EpiDermTM model (tissues were not damaged).

According to the classification criteria given in report, the test substance, Propatyl nitrate, is considered to have no category with regard to skin irritation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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
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
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)
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.
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.
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.

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

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 EpiOcularTM. 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: EpiOcularTMEye 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 EpiOcularTMmodel (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.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.10. – 12. 10. 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Adopted 14th Feb 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic

- Characteristics of donor animals (e.g. age, sex, weight): The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

- Time interval prior to initiating testing: The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). All eyes used in the assay were from the same group of eyes collected on a specific day.

- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects including scratched, and neovascularisation were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.
Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
The test substance was tested neat by direct application onto the corneal surface.


VEHICLE
Hank`s Balanced Salts Solution (HBSS)
- Lot/batch no. (if required): SLBL5152V, Sigma-Aldrich
Duration of treatment / exposure:
4 hrs
Duration of post- treatment incubation (in vitro):
1.5 hr
Number of animals or in vitro replicates:
The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.
Number of corneas per group:
Exposed group (test substance) - 3 corneas (No. 7, 8, 9)
Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas (No. 4, 5, 6)
Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Selection criteria for eyes used in BCOP: Only corneas from eyes free of defects including scratched, and neovascularisation were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.

Preparation: Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium.
Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively.
Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and a baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.
Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

QUALITY CHECK OF THE ISOLATED CORNEAS
From 21 eyes the 2 eyes were eliminated after inductive incubation, because the baseline opacity values were >7.
Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 5, 6, 7, 7 and 9,), 7 eyes was superfluous and remaining 3 were used for the testing of another substance.


NUMBER OF REPLICATES
Number of corneas per group:
Exposed group (test substance) - 3 corneas (No. 7, 8, 9)
Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas (No. 4, 5, 6)
Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)

NEGATIVE CONTROL USED
0.9% NaCl

POSITIVE CONTROL USED
20% Imidazole

APPLICATION DOSE AND EXPOSURE TIME
The open-chamber method was used, because the test substance could not be prepared as a suspension or solution in 0.9% NaCl or water. The test substance (enough to completely cover the cornea) was applied directly to the epithelial surface of the cornea using the spatula. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.

Exposure time: 4 hrs

TREATMENT METHOD: open-chamber method

POST-INCUBATION PERIOD: 1.5 hr

REMOVAL OF TEST SUBSTANCE, POST-EXPOSURE INCUBATION
After the exposure period, the negative control, the positive control substance and the test substance was removed from the anterior chamber with EMEM (containing phenol red
- the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the diminution of lights passing through the cornea. The light was measured as illuminance (l = luminous flux per area, unit: lux) by a light meter.
Corneal opacity (as value of illuminance) was measured quantitatively with the aid of an opacitometer (Opacitometer, Kit OP3,0 – Duratec, Analysertechnik – Germany).

- Corneal permeability: The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
≥ 55 Category 1
Irritation parameter:
in vitro irritation score
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
Study acceptance criteria were fulfilled.

- Acceptance criteria met for negative control: The value of opacity for negative control (0.9% NaCl) obtained during the study was 2.03
and value of permeability was 0.029. The values obtained during this study not exceeded upper limits, so the study is considered acceptable.

- Acceptance criteria met for positive control: The value of IVIS for positive control (20% imidazole) obtained during the study was 78.17.
This value is within the acceptance limit (one standard deviations of the current historical mean), so the study is considered acceptable.

The In Vitro Irritancy Score (IVIS) was computed according the following formula:

IVIS = mean opacity value + (15 x mean permeability OD490 value)

Group

IVIS calculation

Result

NC (0.9% NaCl)

2.03 + 15 x 0.029

2.47

PC (20% Imidazole in 0.9% NaCl)

45.00 + 15 x 2.211

78.17

Test item (Propatyl nitrate)

0 + 15 x 0

0

Interpretation of results:
GHS criteria not met
Conclusions:
The In Vitro Irritancy Score (IVIS) for Propatyl nitrate was 0.
This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.
Executive summary:

The test substance, Propatyl nitrate, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the Method B.47, Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, Adopted 14th February 2017

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Open-chamber method was used, because the test substance was not soluble in water or in 0.9% NaCl solution. The test substance was applied in 100 % concentration. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) is to be calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for Propatyl nitrate was 0.

This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification