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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 Oct 2017 - 21 Nov 2017
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
Qualifier:
according to
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:
26 July 2013
GLP compliance:
yes (incl. certificate)
Remarks:
Országos Gyógyszerészeti és Élelmezés-egészégügyi Intézet

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 38
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Number of animals: not reported
- Characteristics of donor animals: age, sex, and weight not reported
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation by electric current. The heads were transported at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3 - 20.4ºC) during the transport.
- Time interval prior to initiating testing: All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Indication of any existing defects or lesions in ocular tissue samples: The fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit. Selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope with the slit-width set at 9½, equalling 0.095 mm. Any eye with cornea thickness deviating more than 10% from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.03 g
Duration of treatment / exposure:
After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible.
Observation period (in vivo):
The control 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 ± 5 minutes were considered acceptable.
Number of animals or in vitro replicates:
Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Corneas in good condition were selected for examination. Selected eyeballs were carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye 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.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye.

NUMBER OF REPLICATES
Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution.

NEGATIVE CONTROL USED : Saline solution

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED : Imidazole

APPLICATION DOSE AND EXPOSURE TIME : 0.03 g for 10 seconds

OBSERVATION PERIOD : 30, 75, 120, 180, and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature .

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Cornea opacity was measured at all time points by a hand-held slit lamp or slit lamp microscope and observations were scored on a scale from 0-4; the calculation used is Mean CO(at time t) = [FEΔCO (at time t)+ SEΔCO(at time t) + TEΔCO(at time t)]/3 where
CO = Cornea opacity;
ΔCO = Difference between cornea opacity and cornea opacity reference value;
FEΔCO = Difference between first eye cornea opacity and first eye cornea opacity reference value;
SEΔCO = Difference between second eye cornea opacity and second eye cornea opacity reference value;
TEΔCO = Difference between third eye cornea opacity and third eye cornea opacity reference value;
Mean CO = The mean corneal opacity value; at time t = Observation time at 30, 75, 120, 180 or 240 minutes after the post-treatment rinse; and at t=0 =
Reference value

- Damage to epithelium based on fluorescein retention: Fluorescein retention was measured by a hand-held slit lamp or slit lamp microscope and observations were scored on a scale from 0-3; the calculation used is Mean FR = [FEΔFR (at time t) + SEΔFR(at time t) + TEΔFR at time t)]/3 where FR = Fluorescein retention; ΔFR = Difference between fluorescein retention and fluorescein retention reference value; FEΔFR = Difference between first eye fluorescein retention and first eye fluorescein retention reference value; SEΔFR = Difference between second eye fluorescein retention and second eye fluorescein retention reference value; TEΔFR = Difference between third eye fluorescein retention and third eye fluorescein retention reference value; Mean FR = The mean fluorescein retention value; at time t = Observation time at 30 minutes after the post-treatment rinse; and at t=0 = Reference value
- Swelling: swelling was calculated through a calculation CS at time t = (CT at time t – CT at t = 0)/CT at t = 0 X 100 and Mean CS at time t = [FECS(at time t) + SECS (at time t) + TECS (at time t)]/3 where CS = Cornea swelling; CT = Cornea thickness
FECS = First eye cornea swelling; SECS = Second eye cornea swelling; TECS = Third eye cornea swelling; Mean CS = The mean percentage of corneal swelling; at time t = Observation time at 30, 75, 120, 180 or 240 minutes after the post-treatment rinse; and at t = 0 = Reference value

SCORING SYSTEM:
- Mean corneal swelling (%) : no scoring scale was used for mean corneal swelling
- Mean maximum opacity score : 0 = No opacity; 0.5 = Very faint opacity; 1 = Scattered or diffuse areas; details of the iris are clearly visible; 2 = Easily discernible translucent area; details of the iris are slightly obscured; 3 = Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible; 4 = Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment : 0 = No fluorescein retention; 0.5 = Very minor single cell staining; 1 = Single cell staining scattered throughout the treated area of the cornea; 2 = Focal or confluent dense single cell staining; 3 = Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: used criteria established in OECD 438 guideline

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
2.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
75 min
Value:
26
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
240 min
Value:
33
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; see attached document
- Acceptance criteria met for positive control: yes; see attached document

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS Category 1 (H318) according to Regulation (EC) No 1272/2008
Conclusions:
In this eye irritation study (OECD 438), Butylmonoglycol sulphate, Na-salt caused ocular corrosion or severe irritation in the enucleated chicken eyes.