Reaction mass of N-(4-(bis(4-(methylamino)phenyl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate and [4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate and [4-[bis[4-(dimethylamino)phenyl]methylene]-2,5-cyclohexadien-1-ylidene]dimethylammonium acetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

Study following official guidelines performed on a similar substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-[[4-(dimethylamino)phenyl][4-(methylimino)cyclohexa-2,5-dien-1-ylidene]methyl]-N,N-dimethylaniline monohydrochloride

EC Number:

210-042-3

EC Name:

4-[[4-(dimethylamino)phenyl][4-(methylimino)cyclohexa-2,5-dien-1-ylidene]methyl]-N,N-dimethylaniline monohydrochloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Appearance: violet powderStorage: The test substance should be stored in dry room in dark in closed container at the room temperature.Stability / Expiration date: 12/2020

Test system

Vehicle:

other: other: 20% tested material in a 0.9% sodium chloride solution

Details on study design:

Bovine eyes source:Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech RepublicEyes were collected by slaughterhouse employees.The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 60 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 (typically 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.Selection criteria for eyes used in BCOP:The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects 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.From 21 eyes the 6 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 5, 7, 8, 9, 11, 12, 13, 14 and 16,), 3 eyes was superfluous and remaining 3 were used for the testing of another substance.Number of corneas per group:Exposed group (test substance) - 3 corneas (No. 8, 9, 11)Positive control group (20% Imidazole) – 3 corneas (No. 12, 14, 16)Negative control group (0.9% NaCl) – 3 corneas (No. 5, 7, 13)Positive control substance: imidazoleNegative control substance: 0.9% NaClProcedure scheme:Selection of corneas, mounting in holders → incubation with EMEM 1hour (32 ± 1°C) → removed EMEM, measurement of baseline opacity → treatment by positive and negative control substances and test substance (incubation 4 hours) → washing of epithelium, measurement of opacity after application → application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) → measurement of absorbance (490 nm).Preparation of the 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 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.Closed-chamber method was used, because the test substance was applicable by micropipette. The test substance (750 µL of application form) to cover the epithelial side of the cornea is introduced 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. 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).Post-exposure:After the exposure period, the negative control and the positive control 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.The test substance was removed from the anterior chamber with EMEM – repeatedly, because the test substance is coloured . Subsequently the test substance was removed mechanically using a cotton swab and brush. The corneas (applied the test substance) were also rinsed with EMEM (containing phenol red). Lastly EMEM (without phenol red) was used for final rinsing. The test substance was complete removal, but corneas stayed coloured by the test substance. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.Endpoints measured:Opacity: 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.Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC.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.Mean opacity:Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.Mean permeability:Mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean permeability is calculated.IVIS calculation: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 as follows:IVIS = mean opacity value + (15 x mean permeability OD490 value)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

test group

Remarks on result:

not determinable

Remarks:

IVIS could not be calculated for the tested substance since no opacity values could be measured. The opacity after test substance application could not be measured since corneas have been coloured dark violet

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control

Value:

ca. 2.05

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control

Value:

ca. 81.43

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

mean value of opacity differences over corneas # 8,9,11. Test group

Remarks on result:

other: The irritation parameter is cornea opacity. The opacity after test substance application could not be measured since corneas have been coloured dark violet.Opacity differences resulted equal to: cornea #8 : 195, cornea #9: 196 and cornea#11: 196

Remarks:

The irritation parameter is cornea opacity. The opacity after test substance application could not be measured since corneas have been coloured dark violet.Opacity differences resulted equal to: cornea #8 : 195, cornea #9: 196 and cornea#11: 196

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

mean value of opacity differences over corneas # 5,7,13. Negative control

Value:

ca. 1.67

Remarks on result:

other:

Remarks:

The irritation parameter is the corneal difference opacity

Results 6

Irritation parameter:

cornea opacity score

Run / experiment:

mean value of opacity differences over corneas # 12, 14 and 16. Positive control

Value:

ca. 54.97

Remarks on result:

other:

Remarks:

The irritation parameter is the corneal difference opacity

Results 7

Irritation parameter:

fluorescein retention score

Run / experiment:

mean value of permeability differences over corneas n#8, 9 and 11. Test group

Value:

ca. 0.026

Remarks on result:

other:

Remarks:

Irritation parameter is cornea permeability

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

mean value of permeability differences over corneas # 5,7,13, negative control

Value:

ca. 0.025

Remarks on result:

other:

Remarks:

Irritation parameter is cornea permeability

Results 9

Irritation parameter:

fluorescein retention score

Run / experiment:

mean value of permeability differences over corneas # 12, 14 and 16. Positive control

Value:

ca. 1.92

Remarks on result:

other:

Remarks:

Irritation parameter is cornea permeability

Applicant's summary and conclusion

Conclusions:

The substance was tested for eye irritation following OECD 437 in vitro test. Under the experimental conditions the IVIS value could not be calculated. However, the discolouration of the cornea triggers the classification of the substance as corrosive following Regulation 1272/2008

Executive summary:

The substance was tested for eye irritation following OECD 437. Bovine Cornea collected directly from the slaugther were examined for defects and exposed to the tested substance suspended in a 0.9% solution of the tested material (at 20%). Negative and positive control were concurrent with NaCl and imidazole, respectively.The In Vitro Irritancy Score (IVIS) for test substancecould not becalculated. The basic values for the IVIS calculation (opacity values) could not be measured because of colouring of corneas which were applied by the test substance.