Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
Adopted: 29th July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Counsil Regultaion (EC) No. 440/2008, published in O.J.L 142, 2008
Deviations:
not specified
Principles of method if other than guideline:
The test consists of a topical exposure of the test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide] into a blue formazan salt, caused by dehydrogenase present in cell mitochondria. The conversion is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with sterile water for injection) is used to predict the skin corrosion potential.
Effect of a substance is determined by measuring of optical density of the formazan extracts using a spectrophotometer at 570 nm. Relative cell viability is calculated for each triplet of tissues as % of the mean of the negative control tissues. Skin corrosion potential of the test item is predicted according to criteria given in par. 3.9.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch. No.: 23/02/2018
Test system:
human skin model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Cell source:
other: The reconstructed human epidermal model consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis.
Details on test system:
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SK) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ System is manufactured according to defined quality assurance procedures.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing 24 tissues on shipping agarose together with the necessary amount of culture media.
Lot No. of tissues used for this test: 28671.
Control samples:
not specified
Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
other:
Controls:
not specified
Amount / concentration applied:
VEHICLE
- Water for injection, Ardeapharma, Lot No. 1706090516, exp. 06/2019
- PBS (phosphate buffered saline) – prepared in laboratory 29/08/2018, exp. 01/03/2019
- MTT (3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide), Alfa Aesar, Lot No. 10196058, retest date. 2/2026
- Isopropyl alcohol p.a., Lach-Ner, Lot No. PP/2016/04778, exp. 02/2019

NEGATIVE CONTROL
- 50 μL H2O tested with every exposure time

POSITIVE CONTROL
- KOH 8N, solution prepared in laboratory 26/11/2018, exp. 26/05/2019
- 50 μL 8N KOH tested with every exposure

Details on study design:
MTT TEST
- Test item application: 50 μL of the test item was placed directly atop to the tissue and it was spread to match size of the tissue.
- Controls:
Negative control - 50 μL H2O tested with every exposure time
Positive control - 50 μL 8N KOH tested with every exposure time
- Procedure: After delivery, tissues were placed to fresh assay medium and conditioned for about 18 hours to release transport stress related compounds and debris. Next day, tissues were topically exposed to the test
chemicals for 3 (room temperature) and 60 minutes at culture conditions (37±1°C, 5±1 % CO2, humidified. After exposition, tissues were thoroughly rinsed and blotted to remove the test item/controls.
After that, tissues were transferred to 24-well plates containing MTT medium (1 mg*mL-1) and incubated at culture conditions for 3 hours. After MTT incubation, the blue formazan salt (formed by cellular
mitochondria) was extracted with 2.0 mL/tissue of isopropyl alcohol for ca 2 hours at room temperature and shaking. Optical density of the extracted formazan was determined using a spectrophotometer at 570
nm.

OD570 MEASURING
- OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol was used as a blank
Irritation / corrosion parameter:
% tissue viability
Remarks:
after 3 minutes exposure
Run / experiment:
Corrosivity potential of the test materials is predicted from the relative mean tissue viabilities obtained after 3 minutes and 60 minutes treatment compared to the negative control tissues concurrently treated with H2O.
Value:
86.8
Negative controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
after 60 minutes exposure
Value:
1.8
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Tissue was injured after 60 minutes exposure
- Direct-MTT reduction: negative results - not corrected
- Colour interference with MTT: negative results - not correcte


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The absolute OD of the negative control (NC) tissues (treated with sterile water for injection) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing
procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8

- Acceptance criteria met for positive control:
An 8N KOH (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required
per testing day. Viability of positive control should be within 95±1 % confidence interval of the historical data.
The assay meets the acceptance criterion if the mean viability of PC tissues treated for 1 hour expressed as % of the negative control tissues is ≤ 15 %.

- Acceptance criteria met for variability between replicate measurements:
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion
if the coefficient of variance from 3 identically treated tissues is ≤ 0.3. This criterion is additional. At a difference over the limit, the rejection of the outlying tissue should be considered.
Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
Under the above-described experimental design, the test item Pentyl nitrite was corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.
Executive summary:

The test item Pentyl nitrite was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according toOECD Test Guideline 431,In Vitro Skin Corrosion: Human Skin Model Test, Adopted: July 29th, 2016.

Complementary experiments were performed as a part of another study:Study No. 293/18/5AI:Pentyl Nitrite,In vitro Eye IrritationTest (EpiOcularTMmodel); VUOS-CETA Report No.18-436, 2018,both with negative result. Colour of the test item does not interfere with the endpoint and the test item has no direct reduction properties.

Therefore, results of MTT test did not need to be corrected.

           In the MTT test, the test item (50 μL)wasplaced atop the tissue.Length of exposition was 3 and60 minutes. Nine tissues were used for the experiment in each time: three per test item (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.OD570of 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 item Pentyl nitrite was 86.8 % of the negative control average value after 3 minutes treatment and 1.8 % after 60 minutes of treatment.

 

The test item is considered to be corrosive to skin:

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 %.

 

In the experiment arrangement described above, the test item Pentyl nitrite was corrosive in theEpiDermTMmodel.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted 28th February 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23th July 2009
Deviations:
no
Qualifier:
according to
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:
11/7/2017, 1 - 35
Deviations:
no
Principles of method if other than guideline:
The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential.
Viability is determined by measuring of optical density (OD) of the formazan extracts using a spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean OD value of the negative control tissues.
Skin irritation potential of the test substance is predicted if the resulting cell viability is less than or equal (≤) to 50%.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: 23/02/2018
Test system:
human skin model
Source species:
other: The reconstructed human epidermal model consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis.
Cell type:
non-transformed keratinocytes
Cell source:
other: The reconstructed human epidermal model consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis.
Vehicle:
other: PBS (phosphate buffered saline), prepared in laboratory 29/08/2018 exp. 01/03/2019. MTT (3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide), Alfa Aesar, Lot No. 10196058, rec. retest date. 2/2026
Details on test system:
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, Slovakia) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.
The developer/supplier provided demonstration that each batch of the RhE model used meets acceptable production release criteria (Annex 1).
Lot No. of tissues used for experiment was 28671.
Control samples:
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
MTT TEST
- Application: 30 μL of the test item was dosed directly on tissue. The item was spread on the entire tissue surface. A single experiment, composed of three replicate tissues, was run.
- Tissues preparation and treatment:
On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After about 60 minutes of pre-incubation, medium was replaced by a fresh one and pre-incubation continued for other 18±3 hours.
After pre-incubations, tissues were topically exposed to the test chemicals for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Tissues were then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium.
After 24±2 hours of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18±2 hours.

MTT ASSAY:
Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg·mL-1). After 180±5 minutes of MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol (for minimum 120 minutes, room temperature, shaking) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.

OD570 MEASURING:
OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was used.
Type of coverage:
not specified
Preparation of test site:
not specified
Controls:
yes
Irritation / corrosion parameter:
% tissue viability
Value:
<= 80
Negative controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system:
- Direct-MTT reduction: performed with negatice results
- Colour interference with MTT: performed with negative reuslits

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The absolute OD of the negative control (NC) tissues (treated with sterile PBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under
specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8.
The 99% confidence interval of historical negative control is 1.467-2.329

- Acceptance criteria met for positive control:
A 5 % SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is
required per testing day. Viability of the positive control should be within 95±1 % confidence interval of the historical data.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %.
The 99% confidence interval of historical positive control is 0.000-0.185.

- Standard deviation:
Each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, so the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if
the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18 %.
When any of the acceptance criteria is not met, the experiment has to be repeated
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
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 * Category 2.
As the study for corrosivity was performed at the same time (Study No. 293/18/4AC Pentyl nitrite - In vitro Skin Corrosion Test (EpiDermTM Model), VUOS-CETA, Report No. 18-452) with positive result the test item is considered to be corrosive to skin.
Executive summary:

The test item, Pentyl nitrite,was assayed forin vitroskin irritation in the human epidermal model EpiDermTM.The test was performed according totheOECDTest GuidelineNo.439:In Vitro Skin Irritation: Reconstructed HumanEpidermisTest Method(2015) andProtocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

In the complementaryexperiments performed as a part of another study (Study No. 293/18/5AI:Pentyl Nitrite,In vitro Eye IrritationTest (EpiOcularTMmodel); VUOS-CETA, Report No.18-436) either direct MTT reduction or colour interference were not found.Therefore, result of MTT test did not have to be corrected.

In MTT test after pre-incubation of tissues,30 μL of the test itemwasplaced directly on tissue and spread on the entire tissuesurface.The length of exposure was60 minutes. Three tissues were used for the test item and three for positive and negative controls.

After removal of the test item, tissues were post-incubated for approximately
42 hours. Three hours incubation with MTT and extraction period with shaking followed then. 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.

 Under the above-described experimental designaverage viability of treated tissueswas1.0%,i.e. viability was ≤50 %.

The effect of the test itemwaspositiveinEpiDermTMmodel (tissues were damaged).

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 beirritant to skinin accordance with UN GHS * Category 2.

Asthe study for corrosivity was performed at the same time (Study No.293/18/4ACPentyl nitrite-In vitro Skin Corrosion Test(EpiDermTMModel), VUOS-CETA, Report No. 18-452)with positive resultthetest item is considered to becorrosiveto skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Adopted 14th February 2017
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
Adopted 09th October 2017
Deviations:
no
Principles of method if other than guideline:
The BCOP (Bovine Corneal Opacity and Permeability) test method is based on an organotypic model that provides short term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and visible light spectrophotometer, respectively. Both measurements are used to calculate an IVIS (In Vitro Irritancy Score), which is used to assign an in vitro irritancy hazard classification category for prediction of an in vivo ocular irritation of the test item.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 02112018
Species:
other: bovine eyes
Details on test animals or tissues and environmental conditions:
Bovine eyes source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic
Eyes were collected by slaughterhouse employees. 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. 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).
The time interval between collection of the eyes and use of corneas in the BCOP was minimized (collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.
Vehicle:
Hank's balanced salt solution
Controls:
yes
Amount / concentration applied:
Name: Hank`s Balanced Salts Solution (HBSS)
Product No: H1387
Lot no: SLBT4865
Supplier: Sigma-Aldrich
Expiration: 10/2019

Name: Eagle`s Minimum Essential Medium (EMEM)
Product No: M3024
Lot no: SLBV3954
Supplier: Sigma-Aldrich
Expiration: 05/2019

Name: Eagle`s Minimum Essential Medium (EMEM) with phenol red
Product No: M2279
Lot no: RNBG6235
Supplier: Sigma-Aldrich
Expiration: 08/2019

Name: Fluorescein sodium salt
CAS No: 518-44-8
Lot no: SLBL5470V
Supplier: Sigma-Aldrich
Expiration: 11/2023
Duration of post- treatment incubation (in vitro):
After the exposure period, the test item, negative control and positive control were 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 corneas were incubated for an additional two hours at 32 ± 1 ºC with EMEM. At the end of the post-exposure incubation period, the opacity and permeability of each cornea were recorded.
Number of animals or in vitro replicates:
Exposed group (test item) - 3 corneas (No. 8, 9, 10)
Positive control group (100% DMFA) – 3 corneas (No. 4, 6, 7)
Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)
Details on study design:
SCHEME:
Selection of corneas, mounting in holders → incubation with EMEM 1hour (32 ± 1°C) → removed EMEM, measurement of baseline opacity (illuminance) → treatment by positive and negative control substance and test item (incubation 10 min.) → washing epithelium, incubation 2 hour (32 ± 1°C), measurement of opacity (illuminance) after application → application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) → measurement of optical density (490 nm).


EXPERIMENTAL PROCEDUREs:
Preparation of eyes:
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 baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
Each test group (test item, 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.

Application of the test item:

Treatment protocol for semi-solids, creams and waxes was used: The test item was tested undiluted for 10 minutes.
Closed-chamber method was used, because the test item (100% form) was applicable by micropipette. The test substance in amount capable to cover the epithelial side of the cornea (750 µL) was introduced by micropipette into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure.

Control substances:
Concurrent negative controls and positive controls were included in experiment for concrete chemicals see 3.3. The controls were included in the BCOP test method so that nonspecific changes in the test system could be detected and to provide a baseline for the assay endpoints (see chapter 3.3, 3.4). The method of application and amount of the negative and positive control substances was the same as for the test item.


Irritation parameter:
in vitro irritation score
Run / experiment:
Resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group
Value:
100.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Remarks:
0.9% NaCl - 1.04
Positive controls validity:
valid
Remarks:
100% DMFA - 97.75
Irritation parameter:
cornea opacity score
Run / experiment:
the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale.
Value:
ca. 99.39
Vehicle controls validity:
not specified
Negative controls validity:
valid
Remarks:
0.9% NaCl - 0.45
Positive controls validity:
valid
Remarks:
100% DMFA - 94.76
Irritation parameter:
other: Permeability (Optical density values)
Run / experiment:
1 mL sodium fluorescein solution was added to the anterior chamber of the corneal holder, while the posterior chamber is filled with fresh EMEM. The amount of fluorescein that crosses into the posterior chamber was measured by UV/VIS.
Value:
0.094
Vehicle controls validity:
not specified
Negative controls validity:
valid
Remarks:
0.9% NaCl - 0.039
Positive controls validity:
valid
Remarks:
100% DMFA - 0.229
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:

- Acceptance criteria met for negative control:
Mean: 80.73 (obtained from eight measuring)
Standard deviation: 11.56
Upper limit: 103.85
Lowe limit: 57.6
The value of IVIS for positive control (100% DMFA) obtained during the study was 99.39. This value is within the acceptance limit (one standard deviations of the current historical mean), so the study is considered acceptable.

- Acceptance criteria met for positive control:
OPACITY: Mean: 1.60 (obtained from eight measuring), Standard deviation: 0.97, Upper limit: 3.54
PERMEABILITY: Mean: 0.0231 (obtained from eight measures), Standard deviation: 0.0150, Upper limit: 0.0612
The value of opacity for negative control (0.9% NaCl) obtained during the study was 0.45 and value of permeability was 0.039. The values obtained during this study not exceeded upper limits, so the study is considered acceptable.

- Classification according UN GHS:
Decision criteria
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
≥ 55 Category 1

The result of calculation for test item: IVIS = 100.22 . Classification : Category 1
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The In Vitro Irritancy Score (IVIS) for Pentyl nitrite was 100.22. The opacity was detected in corneas treated by the test item after exposure.

This value of IVIS is > 55 therefore the classification of test item effect according to UN GHS criteria for eye irritation or serious eye damage is: Category 1: Serious eye damage.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
other: SOP: EpiOcularTM EIT for the prediction of acute ocular irritation of cemicals, MatTek corp.
Version / remarks:
Version 9, April 6, 2015
Deviations:
no
Principles of method if other than guideline:
The test chemical is applied topically to a minimum of two three-dimensional RhCE tissue constructs and tissue viability is measured following exposure and a post-treatment incubation period.
Some signs of eye injury as corneal opacity, iritis, conjunctival redness and/or conjunctival chemosis, is the result of a cascade of events beginning with penetration of the chemical through the cornea and/or conjunctiva can be not observed in vitro. 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.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
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 distiguish between serious eye damage and eye irritation.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 23/02/2018
- Expiration date of the lot/batch: 30/12/2018
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in tightly closed packaging in well-ventilated spaces at temperature below 15°C. The storeroom must be equipped with a first-aid kit and a source of potable water.
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:
Species:
other: The reconstructed human cornea-like epithelial model EpiOcular TM (OCL-200 ver. 2.0) from MatTek, Bratislava, SK
Details on test animals or tissues and environmental conditions:
The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamos epithelium, morphologicall similar to that found in human cornea. The EpiOcularTM RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showinf a cornea-like structure analogous to that found in vivo.
Lots. No. of tissues used for this test: 27079
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50uL of item/surface ration 39.7 uL/cm2 placed directly atop to the tissue moistened with 20uL of PBS.


CHEMICALS
- PBS (phospate buffered saline)
- prepared in laboratory 29/08/2018 exp. 01/03/2019 (for rinsing of tissues)
- MatTek DPBS 082818ISA exp. 28/08/2019 (for wetting of tissues and for preparation of MTT stock solution)
- MTT (3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide), Alfa Aesar, Lot No. 10196058 recommended retest date 02/2026
- Isopropyl alcohol p.a., Lach-Ner, Lot No. PP/2016/04778, exp. 02/2019

POSSITIVE CONTROL
- Methyl Acetate 99%, MatTek, Lot No. 022118ISA, exp. 21/02/2019

NEGATIVE CONTROL
- Water for injection, Ardeapharma, Lot No. 1706090516, exp. 06/2019

MEDIA
- EPI-100, MatTek, Lot No. 111218ISA, exp. 03/12/2018
Duration of post- treatment incubation (in vitro):
Tissues were incubated for 2 hours ± 15 minutes at standard culture conditions
Details on study design:
A) COMPLEMENTARY EXPERIMENTS:
DIRECT MTT REDUCTION: Some test substances may interfere with the MTT endpoint if they are able to reduce MTT directly. Experiment Direct MTT reduction - functional check in tubes was performed with negative result. Therefore, other step (direct rduction in frozen tissues) did not need to be performed.
COLOUR INTERFERENCE: For non.coloured test items complementary test have to be performed to assess if they become colorants after contact with water or isopropyl alcohol. Test for colour interference was performed with negative result. Therefore, other step (use of colorant control tissues) did not need to be performed.

B) MTT TEST
Two tissues were used per the test item (C3), two for positive (PC) and two for the negative (NC) controls. Procedure is described bellow.
Irritation parameter:
other: average viability of treated tissues
Value:
1.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
not specified
Irritation parameter:
other: viability
Value:
<= 60
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
not specified
Other effects / acceptance of results:
The results are acceptable if:
1) The negative control OD > 0.8 and < 2.5;
2) The mean relative viability of the possitive contol 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 colorand controls which are calculated as percent values related to the viability of the relating negative control.

According to classification criteria, given in Chapter 3.8 of Final report, the test item, Pentyl nitrite, is identified as potentially requiring classification and labelling according to UN GHC (Category 2 or Category 1)

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Remarks:
or Category 1
Conclusions:
Average viability of tissues treated by Pentyl nitrite was 1.4% of negative control average value, i.e. viablitiy was <= to 60%.

The effect of the pentyl nitrite was positive in EpiOcularTM model (tissues were damaged).

According to the classification criteria, pentyl nitrite is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Further testing with other test methods will be required because EpiOcularTM EIT shows a certain number of false results and cannot resolve between UN GHS Categories 1 and 2.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Additional information

Justification for classification or non-classification

The In Vitro Irritancy Score (IVIS) for Pentyl nitrite was 100.22. The opacity was detected in corneas treated by the test item after exposure.

This value of IVIS is > 55 therefore the classification of test item effect according to UN GHS criteria for eyeirritation or serious eye damage is: Category 1: Serious eye damage.

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 * Category 2.

As the study for corrosivity was performed at the same time (Study No. 293/18/4AC Pentyl nitrite - In vitro Skin Corrosion Test (EpiDermTM Model), with positive result the test item is considered to be corrosive to skin.