Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Cas Number:

30199-25-8

Molecular formula:

C11H20O

IUPAC Name:

(4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Constituent 2

Reference substance name:

cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

Cas Number:

69680-38-2

Molecular formula:

C12H24O2

IUPAC Name:

cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

Test material form:

liquid

Details on test material:

- Physical State: Colourless liquid

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS: N/A

Test animals / tissue source

Species:

chicken

Strain:

other: Gallus Gallus e.g. Ross 308 Broiler

Remarks:

Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Baileys Turkeys Ltd., Cheshire, UK
- Age at study initiation: approximately 56 days pre kill.
- Weight at study initiation: 3 kg pre kill.

Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL
- Concentration (if solution): N/A

Duration of treatment / exposure:

See "details on study design"

Observation period (in vivo):

See "details on study design"

Duration of post- treatment incubation (in vitro):

See "details on study design"

Number of animals or in vitro replicates:

The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.

Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are < 0.5.

Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.

Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.

Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.

Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.

EQUILIBRATION AND BASELINE RECORDINGS

After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES

The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED

Sodium chloride solution (0.9 % v/v)

SOLVENT CONTROL USED (if applicable)

Not applicable

POSITIVE CONTROL USED

Benzalkonium chloride solution (5 % v/v)

APPLICATION DOSE AND EXPOSURE TIME

0.03 mL of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds.

OBSERVATION PERIOD

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of isotonic saline
- Indicate any deviation from test procedure in the Guideline: N/A

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item using a Haag-Streit BQ 900 (Switzerland) microscope.
- Swelling: Percentage corneal swelling was assessed from corneal thickness measurements. Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea.
- Macroscopic morphological damage to the surface: Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.
- Others (e.g, histopathology): N/A

SCORING SYSTEM:
- Mean corneal swelling (%): 0 - 5, >5 - 12, >12 - 18 (>75 mins after test item application), >12 - 18 (<75 mins after test item application), >18 - 26, >26 - 32 (>75 mins after test item application), >26 - 32 (<75 mins after test item application), >32
- Mean maximum opacity score: 0 (no opacity), 0.5 (very feint 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 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: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Remarks:

Scattered or diffuse areas; details of the iris clearly visible was noted in all test item treated eyes. Severe corneal opacity/Complete corneal opacity was noted in the positive control treated eyes. Very faint opacity was noted in the negative control treated eyes. No morphological effects were noted in the test item or control item treated eyes. Sloughing was noted in one of the positive control treated eyes.

Results 2

Irritation parameter:

fluorescein retention score

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Remarks:

Very minor single cell staining/Single cell staining scattered throughout the treated area of the cornea. Focal or confluent dense single cell staining/Confluent large areas of the cornea retaining fluorescein were noted in the positive control treated eyes. Very minor single cell staining/No fluorescein retention was noted in the negative control treated eyes.

Results 3

Irritation parameter:

percent corneal swelling

Value:

9.43

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Any other information on results incl. tables

Table 1: Individual Scores and Mean Scores for Corneal Efects - Test Item

End Point	Eye Number	Time (after decontamination) (mins)
		0	30	75	120	180	240
Corneal Opacity	3A	0.0	0.5	1.0	1.0	1.0	1.0
	6A	0.0	0.5	1.0	1.0	1.0	1.0
	8A	0.0	0.5	1.0	1.0	1.0	1.0
Mean		0.0	0.5	1.0	1.0	1.0	1.0
ICE Class		II
Fluoroscein Retention	3A		1.0
	6A		0.5
	8A		1.0
Mean			0.8
ICE Class		II
Corneal Thickness	3A	0.72	0.70	0.74	0.76	0.74	0.74
	6A	0.70	0.76	0.76	0.72	0.74	0.80
	8A	0.70	0.70	0.76	0.77	0.72	0.78
Mean		0.71	0.72	0.75	0.75	0.73	0.77
Mean % Corneal Swelling			1.89	6.60	6.13	3.77	9.43
ICE Class		II
ICE Classes Combined		3 x II
Classification		No prediction for eye irritation can be made.

Mean scores for corneal effects in the positive control group were; corneal opacity (Class IV), fluorescein retention (Class IV), corneal thickness (Class IV). 3 x IV Classes = Category 1 classification.

Mean scores for corneal effects in the negative control group were; corneal opacity (Class I), fluorescein retention (Class I), corneal thickness (Class I). 3 x I Classes = No category classification.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction for eye irritation can be made following assessment of the data for all endpoints.

Executive summary:

The Isolated Chicken Eye (ICE) test was conducted using reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane in accordance with 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" (2013).

Undiluted test item was applied evenly to the surface of three corneas before being washed off with isotonic saline solution after 10 second test item contact time. A negative and positive control group containing 2 and 3 corneas, respectively, were also prepared.

Observations for corneal opacity and corneal thickness were made at 0, 30, 75, 120, 180 and 240 minutes (after rinsing). Fluorescein retention was measured 30 minutes after rinsing.

Corneal opacity, fluorescein retention and corneal thickness were each classified as ICE Class II for the test item group, concluding that no prediction for eye irritation could be made following assessment of the data for all endpoints.