Reaction mass of 2-methylbutyl nitrite and 2-methyl-1-butanol and 1-pentanol and pentyl nitrite

Skin irritation / corrosion

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

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

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

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. QA statement)

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.

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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Adopted: 29th July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methylbutan-1-ol; 2-methylbutyl nitrite; pentan-1-ol; pentyl nitrite

EC Number:

951-169-0

Molecular formula:

C15H46N2O10

IUPAC Name:

2-methylbutan-1-ol; 2-methylbutyl nitrite; pentan-1-ol; pentyl nitrite

Test material form:

liquid

Specific details on test material used for the study:

Batch. No.: 23/02/2018

In vitro test system

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

Test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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.

Applicant's summary and conclusion

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 EpiDerm^TM. The test was performed according toOECD Test Guideline 431,In Vitro Skin Corrosion: Human Skin Model Test, Adopted: July 29^th, 2016.

Complementary experiments were performed as a part of another study:Study No. 293/18/5AI:Pentyl Nitrite,In vitro Eye IrritationTest (EpiOcular^TMmodel); 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 theEpiDerm^TMmodel.