Reaction mass of 1-methyl-4-(1-methylethylidene)cyclohexyl acetate and p-menth-1-en-8-yl acetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

p-menth-1-en-8-yl acetate

EC Number:

201-265-7

EC Name:

p-menth-1-en-8-yl acetate

Cas Number:

80-26-2

Molecular formula:

C12H20O2

IUPAC Name:

1-methyl-1-(4-methylcyclohex-3-en-1-yl)ethyl acetate

Test material form:

liquid

Test animals / tissue source

Species:

other: ROSS, spring chicken eyes

Details on test animals or tissues and environmental conditions:

Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), body weight range approximately 1.5 - 2.5 kg, were used as eye-donors. Heads of these animals were obtained from poultry slaughterhouse v. Miert, Breukelen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 µL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment.

Number of animals or in vitro replicates:

Three test eyes per test sample, one negative control eye and three positive control eyes were selected for testing.

Details on study design:

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2.0% w/v (Minims, Chauvin, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. lf undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (TNO, Zeist, the Netherlands). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10 - 0.15 mL/min (peristaltic pump set at speed 5.00, Watson-Marlow 205C4, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32°C (water pump set at 36.4°C; Lauda 103, Germany). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-streit slit-lamp microscope. Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced. Three test eyes per test sample, one negative control eye and three positive control eyes were selected for testing. Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations. The isolated chicken eyes were exposed to a single application of 30 uL of the test sample for 10 seconds followed by a 20 mL saline rinse. After rinsing, each eye in the holder was returned to its chamber. The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment. Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope. After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at 5 µM and stained with PAS (Periodic Acid-Schiff). The microscopic slides were filed in the archives and kept available for histopathological examination if considered relevant. ln the case of Terpinyl Acetate Alpha, histopathological examination was considered not relevant, because no borderline severe corneal effects were observed.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Value:

42

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

30 min

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

75 min

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

120, 180, 240 min

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

other: chemosis score

Run / experiment:

30 min

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

other: chemosis score

Run / experiment:

75, 120, 180 min

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

other: chemosis score

Run / experiment:

240 min

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

30 min

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Terpinyl Acetate Alpha caused very slight corneal swelling, slight or slight to moderate corneal opacity, and very slight or slight fluorescein retention. The calculated lrritation lndex was 42 (max possible score is 200). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control eyes showed severe corneal effects and demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants.

Applicant's summary and conclusion

Interpretation of results:

other: Not irritating

Remarks:

in accordance with EU CLP (EC No. 1272/2008 and its updates)

Conclusions:

Terpinyl Acetate Alpha is not an eye irritant /serious eye irritation or serious eye damage in the isolated chicken eye test (OECD guideline 438).

Executive summary:

In an isolated chicken eye test performed according to OECD TG 438 and in compliance with GLP, the eye irritation potential of Terpinyl-Acetate-Alpha was evaluated. Isolated chicken eyes were exposed to a single application of 30 uL of the test sample for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. Terpinyl Acetate Alpha caused very slight swelling of the cornea, slight or slight to moderate corneal opacity, and very slight or slight fluorescein retention. The calculated lrritation lndex was 42 (max possible score is 200). The negative control (saline) caused no corneal effects. The positive control BAC 5% caused moderate corneal swelling, severe opacity and severe fluorescein retention. The calculated lrritation lndex was 42 (max possible score is 200). Terpinyl Acetate Alpha is not an eye irritant /serious eye irritation or serious eye damage in the isolated chicken eye test.