1-(3-sulphonatopropyl)-2-vinylpyridinium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-13 - 2017-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 438 (2013): “Isolated chicken eye test method for identifying ocular corrosives I) chemicals inducing serious eye damage and severe irritants II) chemicals not requiring classification for eye irritation or serious eye damage”.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

EU Method B.48.: “Isolated chicken eye test method for identifying ocular corrosives and severe irritants” Commission Regulation (EU) No. 1152/2010 of December 08, 2010.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(3-sulphonatopropyl)-2-vinylpyridinium

EC Number:

229-565-3

EC Name:

1-(3-sulphonatopropyl)-2-vinylpyridinium

Cas Number:

6613-64-5

Molecular formula:

C10H13NO3S

IUPAC Name:

3-(2-vinylpyridinium-1-yl)propane-1-sulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored at room temperature (20± 5ºC). It was protected from exposure light, humidity and water.

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Chicken were killed for consumption in a licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice.
- Characteristics of donor animals (e.g. age, sex, weight): Eyes used in the study were obtained from healthy approximately 7-week-old chickens whose body weight ranged from 1.5 to 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After sedation of the chickens by electric shock and incision of the neck for bleeding, their heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container. The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 45 minutes. All eyeballs used in the test came from the same group of eyes collected on a specific day.
- Time interval prior to initiating testing: 45 min
- indication of any existing defects or lesions in ocular tissue samples: After a careful excision of the eyelids so as not to damage the cornea, the eyeballs were checked for the presence of the damage. The surface of the eyeballs was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED (HAAG STREIT) to ensure that the cornea was undamaged. Only eyeballs without damage were dissected.
- Indication of any antibiotics used: none stated

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g (test item / positive control) / 0.03 ml (negative control)

Duration of treatment / exposure:

The test item and the control items were uniformly applied to the corneal surface for 10 seconds. Then, they were rinsed from the eye with 20 mL of physiological saline at ambient temperature.

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

The corneas treated with the test item and the control items were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

Number of animals or in vitro replicates:

3 replicates / test item or controls

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After a careful excision of the eyelids so as not to damage the cornea, the eyeballs were checked for the presence of the damage. The surface of the eyeballs was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED (HAAG STREIT) to ensure that the cornea was undamaged. Only eyeballs without damage were dissected.

EQUILIBRATION AND BASELINE RECORDINGS
Prior to the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32°C ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. During the incubation, the eyeballs were continuously supplied with physiological saline at constant temperature of 33 ± 0.5°C and in the average volume of 0.10-0.15 mL/min. After that, a zero reference measurement for corneal thickness and opacity was recorded. It served as a baseline (i.e. time = 0). The fluorescein score determined at dissection of eyeball served as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3 per test item or controls

NEGATIVE CONTROL USED: physiological saline was applied in a volume of 0.03 mL.

POSITIVE CONTROL USED: 0.03 g imidazole

APPLICATION DOSE AND EXPOSURE TIME: 0.03 g / 0.03 ml; the test item and the control items were uniformly applied to the corneal surface for 10 seconds.

OBSERVATION PERIOD: The corneas treated with the test item and the control items were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: They were rinsed from the eye with 20 mL of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: none stated

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by assigning appropriate values to opaque areas. The mean corneal opacity value for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, the final category score for the test item and the control items was given. The corneal opacity scores are given in the guideline. Results of corneal opacity for all examined eyeballs were less than 0.5.
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention value for all test eyeballs was calculated on the grounds of the observations made 30 minutes after the end of the exposure. This value was used to determine the general category score for the test item and the control items. The fluorescein retention scores are given in the guideline. Results of fluorescein retention for all examined eyeballs were less than 0.5.
- Swelling: Deviation of corneal thickness for all examined eyeballs was less than 10%. The corneal thickness was determined using of the depth measuring device no. 1 on the slit-lamp microscope and using of SP-100 pachymeter (TOMEY). The slit-width on slit-lamp microscope was set at 0.095 mm. The pachymeter measures the period of time from emitting ultrasounds to receiving the same ultrasound, reflected by corneal posterior part. Applying a calculation formula shown below, corneal thickness was calculated. L=V x t/2, where L- corneal thickness; V-ultrasound velocity trough cornea; t-measured time.
Measured with optical pachymeter on a slit-lamp microscope; slit-width setting: The degree of corneal swelling was determined by measuring corneal thickness using a slit-lamp microscope and a pachymeter (30 minutes after treatment only pachymeter was used). It was expressed as a percentage and calculated according to the following formula: corneal swelling = (corneal thickness at time t – corneal thickness at time t 0 / corneal thickness at time t 0) * 100. The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, the final category score for the test item and the control items was given.
- Macroscopic morphological damage to the surface: The aim of gross evaluation was to determine whether any morphological effects, e.g. pitting of corneal epithelial cells, roughening of the corneal surface, and sticking of the test item to the cornea were visible.
- Others (e.g, histopathology): Following the final evaluation of the treated eyeballs (240 minutes after the application of the test item and the control items), the eyeballs were fixed in a 4% solution of formaldehyde for at 24 hours. Next, specimens were collected (one specimen in the plane including the cornea, lens, and optic nerve). The tissue material was dehydrated (2 hours in a mixture of 4% phenol/70% ethanol; 2 hours in 95% ethanol; 3 hours in 100% ethanol; 2 hours in equal volumes of xylene and 100% ethanol; 4 hours in xylene) and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using Hematoxylin and Eosin. The histological preparations were evaluated under a light microscope. The following layers of the cornea were evaluated: the anterior corneal epithelium, the anterior elastic lamina (Bowman’s membrane), the corneal stroma, the posterior elastic lamina (Descemet’s membrane), the posterior corneal epithelium. All treated eyeballs were subject to this evaluation.

SCORING SYSTEM:
- Mean corneal swelling (%): As set out in the Guideline.
- Mean maximum opacity score: As set out in the Guideline.
- Mean fluorescein retention score at 30 minutes post-treatment: As set out in the Guideline.

DECISION CRITERIA: decision criteria as indicated in the TG was used.
The results from fluorescein retention corneal opacity, swelling were evaluated separately in order to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined in order to generate an GHS Classification for eyes in vitro for the test item and the control items. In the case of solid materials leading to a GHS “No Category” outcome, a second run of three eyes should be performed to confirm or discard the negative outcome. The second run was not necessary. The test can be considered acceptable if the values obtained in the concurrent positive and negative controls fall within the acceptable ranges for this method. The positive control should be identified as GHS Category 1 and the negative control as GHS Non-Classified.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

SPV, 0 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class I

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

SPV, 30 min

Value:

2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class IV

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

SPV, 0 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class I

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

SPV, 30 min

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class II

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

SPV, 75 min

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class III

Results 6

Irritation parameter:

cornea opacity score

Run / experiment:

SPV, 120, 180, 240 min

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class III

Results 7

Irritation parameter:

percent corneal swelling

Run / experiment:

SPV, 0 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class I

Results 8

Irritation parameter:

percent corneal swelling

Run / experiment:

SPV, 30 min

Value:

-5.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class I

Results 9

Irritation parameter:

percent corneal swelling

Run / experiment:

SPV, 75 min

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class I

Results 10

Irritation parameter:

percent corneal swelling

Run / experiment:

SPV, 120 min

Value:

5.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class II

Results 11

Irritation parameter:

percent corneal swelling

Run / experiment:

SPV, 180 min

Value:

13.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class II

Results 12

Irritation parameter:

percent corneal swelling

Run / experiment:

SPV, 240 min

Value:

18.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

ICE class III

Results 13

Irritation parameter:

morphological effects

Run / experiment:

SPV, 0 - 240 min

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

No changes were observed

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The study was conducted under GLP according to OECD guideline 438 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the eye in vitro.
On the grounds of the study results described in this Report and the overall in vitro Irritancy Classification, it may be stated that the test item, i.e. 1-(3-Sulfopropyl)-2-vinyl-pyridinum betain (SPV) probably did not cause serious eye damage, but it can have negative effect on cornea. According to UN GHS classification criteria no prediction can be made, since the ICE Class combination of the 3 endpoints were 1 x IV, 2 x III. On the basis of the results obtained in the course of the histopathological evaluation it can be concluded that the test item can have a negative effect on the chicken cornea in the ICE test, however the test item cannot be put into category 1.
Hence, no conclusion can be drawn whether the substance needs to be classified as GHS category 1 or 2, for a clear distinction an in vivo study would be required based on the current state of the art. However, based on the tonnage band of the substance, in vivo testing is not required, and with regard to animal welfare no further testing will be performed and the result of the present study will be considered inconclusive.

Executive summary:

Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classifications for Eye Irritation or Serious Eye Damage was performed according to OECD 438 under GLP in order to obtain information on health hazards resulting from a superficial influence of the test item, i.e. 1-(3-Sulfopropyl)-2-vinyl-pyridinum betain (SPV), on the eye.

In the isolated chicken eye test (in vitro), toxic effects to the cornea were measured by a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention), a qualitative assessment of opacity, a quantitative measurement of increased thickness (swelling), and qualitative macroscopic and histopathological evaluations of morphological damage to the surface.

The study was conducted on nine eyeballs. The second run was not necessary.

In order to control the quality of the procedure, the eyeballs used for the purpose of the experiment were assessed for potential damage by corneal opacity profusion and fluorescein retention determination. Result of corneal opacity and fluorescein retention for all examined eyeballs was less than 0.5. Deviation of corneal thickness for all examined eyeballs was less than 10%. Before the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32 ± 1.5°C for 45-60 minutes in the superfusion apparatus in order to equilibrate them to the test system. After that, a zero reference measurement for corneal thickness and opacity was recorded. The test item (ground to a powder) and the item used in the positive control (imidazole) were applied in the amount of 0.03 g, whereas the item used in the negative control (physiological salt) was applied in a volume of 0.03 mL. Three eyeballs were used for the test item and three for each control item. Every time, the test item and the control items were applied to the corneal surface for 10 seconds and kept at temperature between 20一23° C. Then, they were rinsed from the eye with 20 mL of physiological salt solution at ambient temperature.

Next, the eyeballs in their holders were placed in the superfusion apparatus in the original upright position.

The corneas treated with the test item and the control items were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure.

Following the final evaluation of the treated eyeballs, i.e. 240 minutes after the application of the test item and the control items, the eyeballs were fixed in a 4% solution of formaldehyde in order to allow histopathological examinations to be conducted.

Results of the studies:

For eyeballs treated with the test item:

the mean fluorescein retention value was equal to 2.7 (ICE class IV),

the maximal mean corneal opacity value was equal to 2.0 (ICE class III),

the maximal mean corneal swelling value was equal to 18.8 % (ICE class III),

no changes of the corneal surface were observed

histopathological examinations revealed: cell vacuolation and necrosis of the superficial and basal layer and slight erosions of the superficial layer of the anterior corneal epithelium (eyeball no. 1), necrosis and erosions of the superficial layer of the anterior corneal epithelium (eyeball no. 2), necrosis and erosions of the superficial and wing layers of the anterior corneal epithelium (eyeball no. 3), local dissection of the corneal stroma (eyeball no. 1), dissection of the corneal stroma (eyeballs no. 2, no. 3).

These results can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method.

Interpretation of the study results:

For test item (1-(3-Sulfopropyl)-2-vinyl-pyridinum betain (SPV)) the prediction cannot be made according to UN GHS classification criteria (1 x IV, 2 x III).