N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-03-24 to 2020-05-???

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

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:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

EC Number:

850-698-3

Cas Number:

2387913-24-6

Molecular formula:

not applicable

IUPAC Name:

N,N,N-tri-C8-C10-alkyl-N-methyl-1-aminium sulfate

Test material form:

other: solified melt

Details on test material:

- Name of test material ((as cited in study report):
Product name: V10069,
Systemic name: Quaternary ammonium compounds, tri-C8-10-alkylmethyl-methylsulfate

Specific details on test material used for the study:

The test item was applied in its original form, considered as 100% (UVCB).

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni utca 129., Hungary)

Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Charles River Laboratories Hungary Kft. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied in its original form.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
The test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea. As the test item was sticky, an alternative treatment way was used: a thin even layer of test item (approximately 0.5 g) was spread on a disk of Parafilm and then placed onto the cornea surface to ensure a suitable exposure. The amount of the test item was enough to adequately expose the entire surface of the cornea.

Duration of treatment / exposure:

10 s

Observation period (in vivo):

eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after the post-treatment rinse

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

Details on study design:

TEST PROCEDURE

Treatment:

After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea. As the test item was sticky, an alternative treatment way was used: a thin even layer of test item (approximately 0.5 g) was spread on a disk of Parafilm and then placed onto the cornea surface to ensure a suitable exposure. The amount of the test item was enough to adequately expose the entire surface of the cornea.

The positive control eyes were treated in a similar way with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution).

Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.


Test item removal:

The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.

Additional gentle rinsing with 3x20 mL saline was performed at each time point when the test material or the positive control material remaining on the cornea was observed.

Note: Physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Lot number: 194948162, Expiry date: 30 November 2022) was used for rinsing.
 

OBSERVATION

Observation and assessment of corneal effects:

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

100%, up to 75 min

Value:

8.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

100%

Value:

3.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

100%

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

percent corneal swelling

Run / experiment:

100%, up to 240 min

Value:

23.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

TEST ITEM

Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 8.8 % II
Mean maximum corneal swelling at up to 240 min 23.6 % III
Mean maximum corneal opacity change 3.67 IV
Mean fluorescein retention change 3.00 IV
Other Observations Slight loosening of epithelium was observed in one eye at 240 minutes and severe loosening of epithelium was observed in one eye at 120 minutes after the post-treatment rinse. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class 1xIII 2xIV

Based on this in vitro eye irritation study in isolated chicken eyes with PC-2019-853, the test item was classified as severe irritant according to the EU regulations. GHS Classification: Category 1.


POSITIVE CONTROL

Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 9.2% II
Mean maximum corneal swelling at up to 240 min 20.1% III
Mean maximum corneal opacity change 4.00 IV
Mean fluorescein retention change 3.00 IV
Other Observations Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the
post-treatment rinse.
Overall ICE Class 1xIII 2xIV

Based on these observations, the positive control substance Imidazole was classified as severe irritant according to the EU regulations. GHS Classification: Category 1.


NEGATIVE CONTROL

Observation Value ICE Class
Mean maximum corneal swelling at up to 75 min 0.0% I
Mean maximum corneal swelling at up to 240 min 0.0% I
Mean maximum corneal opacity change 0.00 I
Mean fluorescein retention change 0.00 I
Other Observations None
Overall ICE Class 3xI

The negative control Physiological saline was classified as non-irritating, according to the EU regulations. GHS Classification: No Category.


VALIDITY OF THE TEST

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Any other information on results incl. tables

SUMMARY TABLE FOR UN GHS CLASSIFICATION

 

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:	False
No severe corneal morphological changes:	False
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:	False

 

Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	True
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	True
Severe loosening of epithelium (in at least 1 eye):	True

 

Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	True

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on this in vitro eye irritation assay in isolated chicken eyes with PC-2019-853, the test item was severely irritant, UN GHS classification: Category 1.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD
No. 438 guideline (25 June 2018).

 

After the zero reference measurements, the eye was held in horizontal position and 0.5 g oftest itemwas applied onto the centre of the cornea such that the entire surface of the cornea was covered (due to the physical nature of the test item an alternative treatment way was used). After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with test item. The three positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

 

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the study was considered to be valid.

Moderate corneal swelling change (mean = 23.6%) was observed during the four-hour observation period on test item treated eyes. Severe cornea opacity change (severity 3 on one eye and severity 4 on two eyes) was observed. Severe fluorescein retention change (severity 3) was noted on three eyes.Slight loosening of epithelium was observed in one eye at 240 minutes and severe loosening of epithelium was observed in one eye at 120 minutes after the post-treatment rinse.Test item was stuck on all cornea surfacesafter the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.

Based on this in vitro eye irritation assay in isolated chicken eyes with PC-2019-853, the test item was severely irritant,UN GHSclassification: Category 1.