Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-4-isopropenyl-1-methylcyclohexene

Constituent 2

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

Test material form:

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: ca. 2 - 8ºC in the dark and under nitrogen
- Stability under test conditions: Asumed 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: No
- Preliminary purification step (if any): No
- 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): n/a - applied unchanged as supplied

OTHER SPECIFICS: No

Test animals / tissue source

Species:

chicken

Strain:

other: Ross 308 Broiler

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Baileys Turkeys Ltd., Cheshire, UK
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): 3 kg and 56 days old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported in boxes containing moistened paper towels (isotonic saline) at ambient temperature.
- Time interval prior to initiating testing: Not reported
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

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 mL
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

Duration of treatment / exposure:

240 mins

Observation period (in vivo):

30, 75, 120, 180 and 240 mins (± 5 mins) after the eyes were decontaminated with isotonic saline (i.e. removal of test item).

Number of animals or in vitro replicates:

3 replicates per test group

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

0.9 % Sodium chloride solution

SOLVENT CONTROL USED (if applicable)

n/a

POSITIVE CONTROL USED

5 % benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME

0.03 mL 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: Treatments rinsed from the eye using 20 mL of isotonic saline.
- Indicate any deviation from test procedure in the Guideline: No

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.
- Swelling: 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. 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
- 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): No

SCORING SYSTEM:
- Mean corneal swelling (%): ((corneal thickness at time (t) - corneal thickness at t0) / corneal thickness at t0) * 100
- Mean maximum opacity score: Scoring from 0 - 4
- Mean fluorescein retention score: Scoring from 0 - 3

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

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximal

Value:

6.05

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non-Classified and GHS Category 1, respectively.

In vivo

Other effects:

No morphological effects were noted in the test item or control item treated eyes. Sloughing was noted in all positive control treated eyes.

Any other information on results incl. tables

Table 1       Individual scores and mean scores for corneal effects - test item

Endpoint	Eye #	Time (after eye rinsng) in mins
		0	30	75	120	180	240
Corneal opacity	3B	0.5	0.5	0.5	0.5	0.5	0.5
	6B	0	0.5	1	2	2	2
	8B	0.5	0.5	0.5	0.5	0.5	0.5
Mean		0.3	0.5	0.7	1.0	1.0	1.0
ICE Class		II
Fluorescein retention	3B	-	0	-	-	-	-
	6B	-	0.5	-	-	-	-
	8B	-	0	-	-	-	-
Mean		-	0.2		-	-	-
ICE Class		I
Corneal thickness	3B	0.72	0.70	0.72	0.76	0.74	0.76
	6B	0.70	0.74	0.78	0.75	0.76	0.76
	8B	0.73	0.70	0.70	0.70	0.72	0.76
Mean		0.72	0.71	0.73	0.74	0.74	0.76
Mean Corneal Swelling (%)			-0.47	2.33	2.79	3.26	6.05
ICE Class		II
ICE Classes Combined		1 x I, 2 x II
Classification		No prediction can be made

Table 2       Individual scores and mean scores for corneal effects - positive control

Endpoint	Eye #	Time (after eye rinsng) in mins
		0	30	75	120	180	240
Corneal opacity	2B	0	3	4	4	4	4
	5B	0.5	4	4	4	4	4
	7B	0	.	.	4	4	4
Mean		0.2	3.3	3.7	4.0	4.0	4.0
ICE Class		IV
Fluorescein retention	2B	-	3	-	-	-	-
	5B	-	3	-	-	-	-
	7B	-	3	-	-	-	-
Mean			3.0		-	-	-
ICE Class		IV
Corneal thickness	2B	0.72	0.94	0.90	0.92	0.84	1.08
	5B	0.70	0.88	0.96	0.96	1.04	1.12
	7B	0.70	0.82	0.84	0.90	0.98	1.06
Mean		0.71	0.88	0.90	0.93	0.95	1.09
Mean Corneal Swelling (%)		-	24.53	27.36	31.13	34.91	53.77
ICE Class		IV
ICE Classes Combined		3 x IV (sloughing in all eyes)
Classification		GHS Category 1

Table 3       Individual scores and mean scores for corneal effects - negative control

Endpoint	Eye #	Time (after eye rinsng) in mins
		0	30	75	120	180	240
Corneal opacity	1B	0	0	0.5	0.5	0.5	0.5
	4B	0	0	0.5	0.5	0.5	0.5
Mean			0	0	0.5	0.5	0.5
ICE Class		I
Fluorescein retention	1B	-	0	-	-	-	-
	4B	-	0	-	-	-	-
Mean		-	0.0		-	-	-
ICE Class		I
Corneal thickness	1B	0.72	0.66	0.73	0.68	0.70	0.72
	4B	0.70	0.70	0.72	0.70	0.74	0.70
Mean		0.71	0.68	0.73	0.69	0.72	0.71
Mean Corneal Swelling (%)		-	-4.23	2.11	-2.82	1.41	0.00
ICE Class		I
ICE Classes Combined		3 x I
Classification		No prediction can be made

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:

OECD 438 (2017) - The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye.

0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with saline for control purposes.

 

After 240 mins incubation (post rinsing) the mean corneal opacity of the chicken eyes was scored as 1.0 (ICE Class II), after 30 minutes the mean fluorescein retention scored as 0.2 (ICE Class I); and after 240 mins mean maximal corneal thickness scored as +6.05 % (ICE Class II).

 

The combination of the three endpoints resulted in an overall ICE score of 1 x I and 2 x II, thereby no classification for eye irritation could be made.