3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2018 (Experimental Start) to 22 January 2019 (Experimental Completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

none

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

EC Number:

230-512-1

EC Name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Cas Number:

7158-25-0

Molecular formula:

C15H18

IUPAC Name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Materia Inc.
- Lot/batch No.of test material: RP229-0714
- Expiration date of the lot/batch: 06 June 2020
- Purity test date: 28 March 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70% Relative Humidity), under inert (N2) gas.
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: Non-soluble in physiological saline. The test item was applied in its original form, as supplied.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No, test item applied in its original form, as supplied.

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Not specified

Test animals / tissue source

Species:

chicken

Strain:

other: Ross 308

Remarks:

Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út. 129., Hungary

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

Test item applied in its original form.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): In each experiment, as the test item was waxy, an alternative treatment method was used: a thin even layer of test item (ca. 1 g) was spread on a disk of Parafilm and then placed onto the cornea surface to ensure suitable exposure. The amount of test item was enough to adequately expose the entire surface of the cornea.

- In each experiment one negative control eye was treated with 30 µL of physiological saline. An additional negative control eye (“Parafilm control”) was treated using the alternative treatment method to show it had no impact on the results.
- In each experiment positive control eyes were treated with 30 mg powdered Imidazole.

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The time of application was noted, then after an exposure period of 10 seconds from the end of 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.

Additional gentle rinsing with at least 20 mL saline was performed at each time point (30, 75, 120, 180 and 240) when the test item or positive control material remaining on the cornea was observed.

Number of animals or in vitro replicates:

In each experiment, three test item treated eyes, three positive control treated eyes, one negative control and one parafilm control treated eye were examined.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eyes selection: On receipt the chicken head was placed 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. The fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

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

The prepared eye was placed in a steel clamp, avoiding too much pressure on the eye, with the cornea positioned vertically (i.e. in the same position as in the chicken head). Due to the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube (at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes). The chamber door was closed with the exception of any subsequent manipulation or examinations, to maintain temperature and humidity.

Eyes examination and acclimatization time: The appropriate number of eyes were selected and placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure they were in good condition. The focus was adjusted to clearly see the physiological saline 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, 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. If the selected eyes were appropriate for the test, then acclimatization was started and conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at a controlled temperature (32 ±1.5°C) during the acclimatization and treatment periods.

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. The cornea thickness should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in experiments. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered suitable for assay use.

NUMBER OF REPLICATES : In each experiment, three test item treated eyes, three positive control treated eyes, one negative control and one "parafilm control" treated eye were examined.

NEGATIVE CONTROL USED : 30 µL of physiological saline (0.9% (w/v) NaCl solution)

An additional control eye (i.e. parafilm control, using one eye) was also run in conjunction with the test item to show that the alternative treatment method (as the test item was waxy, due to the physical nature of the test item an alternative treatment method of treatment was used) had no impact on the results.

POSITIVE CONTROL USED : 30 mg powdered Imidazole.

APPLICATION DOSE AND EXPOSURE TIME : Ca.1g of test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was uniformly covered (due to the physical nature of the test item an alternative treatment method was used). After 10 seconds, the surface was rinsed with 20 mL physiological saline solution at ambient temperature.

OBSERVATION PERIOD : The control eyes and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Post exposure and rinseing, an additional gentle rinsing with at least 20 mL saline was performed at each time point (30, 75, 120, 180 and 240) until all residual test item or positive control material on the cornea had been removed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and corneal opacity were measured at all time points. A Haag-Streit Bern 900 slit-lamp microscope was used for measurements.



Corneal swelling was calculated according to the following formulae:
CS (Corneal swelling) at time t = CT (Corneal thickness) at time t –CT at t=0 / CT at t=0 (x100)

Mean CS at time t = FECS(at time t) + SECS(at time t) + TECS(at time t) / 3

FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point

- Damage to epithelium based on fluorescein retention: The cornea thickness was measured, 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. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. A Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.


- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting:
Small negative numbers for swelling (0 to -5%) following application are evaluated as class I.
Large negative numbers (>12% below control) are probably due to erosion and indicate a severe effect (scored as class IV).
Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).


- Macroscopic morphological damage to the surface: None
- Mean maximum opacity score : See below tables for ICE classification criteria for corneal thickness and opacity:
- Mean fluorescein retention score at 30 minutes post-treatment : See below table for ICE classification criteria for mean fluorescein retention

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. yes

UN GHS Classification Combinations of the three ICE Classes
No Category 3×I
2×I, 1×II
1×I, 2×II
No prediction can be made Other combinations

Category 1 3×IV
2×IV, 1×III
2×IV, 1×II*
2×IV, 1×I*
Corneal opacity = 3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of epithelium (in at least 1 eye)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 1 & 2 (Physiological saline)

Value:

0

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 1 & 2 (Physiological saline)

Value:

0

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 1 & 2 (Physiological saline)

Value:

0

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 1 & 2 (Parafilm control)

Value:

0

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 1 & 2 (Parafilm control)

Value:

0

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 1 & 2 (Parafilm control)

Value:

0

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 1 (Distilled TCPD)

Value:

1.1

Remarks on result:

no indication of irritation

Results 8

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 2 (Distilled TCPD)

Value:

1.6

Remarks on result:

no indication of irritation

Results 9

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 1 (Distilled TCPD)

Value:

0.5

Remarks on result:

no indication of irritation

Results 10

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 2 (Distilled TCPD)

Value:

0.5

Remarks on result:

no indication of irritation

Results 11

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 1 (Distilled TCPD)

Value:

1

Remarks on result:

no indication of irritation

Results 12

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 2 (Distilled TCPD)

Value:

0.83

Remarks on result:

no indication of irritation

Results 13

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 1 (Imidazole)

Value:

24.9

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 14

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment 2 (Imidazole)

Value:

26.1

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 15

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 1 (Imidazole)

Value:

4

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 16

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment 2 (Imidazole)

Value:

4

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 17

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 1 (Imidazole)

Value:

3

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 18

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment 2 (Imidazole)

Value:

3

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: The chosen test facility has trained staff technicallly proficient in this OECD 438 test. It is a GLP compliant facility and has adequate historical data to justify the observed results.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: As per OECD guidence.

Any other information on results incl. tables

SUMMARY TABLE FOR UN GHS CLASSIFICATION (EXPERIMENT I & 2)

 

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

 

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

 

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:	False

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these in vitro eye irritation assays in isolated chicken eyes with Distilled Tricyclopentadiene, the test item was non-irritant, UN GHS Classification: No Category.

Executive summary:

Based on these in vitro eye irritation assays in isolated chicken eyes with Distilled Tricyclopentadiene, the test item was non-irritant, UN GHS Classification: No Category.

Experiment I: No significant swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on all three eyes. Slight fluorescein retention change (severity 1) was observed on all three eyes. Residual test item was stuck on one cornea surface after the post-treatment rinse, this was cleared at 30 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on all three eyes. Slight fluorescein retention change (severity 0.5 on one eye and severity 1 on two eyes) was observed on all three eyes.

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