α,2,2,6-tetramethylcyclohexene-1-butyraldehyde

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 10th, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

α,2,6,6-tetramethyl-2-cyclohexene-1-butyraldehyde

EC Number:

277-458-5

EC Name:

α,2,6,6-tetramethyl-2-cyclohexene-1-butyraldehyde

Cas Number:

73398-85-3

Molecular formula:

C14H24O

IUPAC Name:

2-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butanal

Constituent 2

Reference substance name:

α,2,6,6-tetramethylcyclohexene-1-butyraldehyde

EC Number:

282-993-2

EC Name:

α,2,6,6-tetramethylcyclohexene-1-butyraldehyde

Cas Number:

84518-22-9

Molecular formula:

C14H24O

IUPAC Name:

2-methyl-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butanal

Specific details on test material used for the study:

Test material name (as stated in the report): Cetonal
Batch No.: SC00011028
Expiry date: 23 June, 2016

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The eyes collected from Chickens obtained from a slaughterhouse where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse. (approximately 7 weeks old 1.5 - 2.5 Kg).
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding . Because eyes were dissected in the laboratory , the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with physiological saline.

The eyelids are carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull taking care not to damage the cornea. The eyeball is pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles are cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye is placed on an absorbent pad and the nictitating membrane and other connective tissue are cut away.

Test system

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

30 microL of test item was applied as supplied to the cornea such taht the entire surface of the cornea is evenly covered with the test item.

Duration of treatment / exposure:

Test item was applied for 10 seconds and then rinsed from the eye with 20 mL of physiological saline at ambient temperature. As residual test item was observed on the cornea, four rinses of 10 mL of physiological saline were added.

Duration of post- treatment incubation (in vitro):

Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180 and 240 minutes after the post treatment rinse.

Number of animals or in vitro replicates:

3 enucleated chicken eyes

Details on study design:

Damages by the test substance were assessed by determination of corneal swilling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean score

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class II

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score

Value:

0.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class II

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

maximal mean score

Value:

32

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 240 post dose, corresponding to ICE class III

Other effects / acceptance of results:

The combination of the three endpoints for the positive control, 5% Benzalconium chloride was 3 x IV. Therefore, the positive controle is classified as "Corrosive/Severe irritant" as expected.
The combination of the three endpoints for the negative control, physiological saline was 3 x I. Therefore, the positive controle is classified as "No category" as expected.

Any other information on results incl. tables

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 1.0 corresponding to ICE class II

- mean score of fluorescein retention: 0.7 corresponding to ICE class II

- maximal mean corneal swelling : + 32 at 240 minutes post dose corresponding to ICE class III

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained, under this experimental conditions enable to conclude that the test item Cetonal does not have to be classified in category 1 "irreversible effects on eye".

Executive summary:

This experience on Cetonal has been performed according to the OECD guideline No 438 adopted 26 July 2013.

The eyes collected from Chickens obtained from a slaughterhouse where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse. (approximately 7 weeks old 1.5 - 2.5 Kg). Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding . Because eyes were dissected in the laboratory , the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with physiological saline. The eyelids are carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull taking care not to damage the cornea. The eyeball is pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles are cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye is placed on an absorbent pad and the nictitating membrane and other connective tissue are cut away. 30 microL of test item was applied as supplied to the cornea such taht the entire surface of the cornea is evenly covered with the test item. Test item was applied for 10 seconds and then rinsed from the eye with 20 mL of physiological saline at ambient temperature. As residual test item was observed on the cornea, four rinses of 10 mL of physiological saline were added. Damages by the test substance were assessed by determination of corneal swilling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The ocular reactions observed in eyes treated with the test item were: - maximal mean score of corneal opacity: 1.0 corresponding to ICE class II - mean score of fluorescein retention: 0.7 corresponding to ICE class II - maximal mean corneal swelling : + 32 at 240 minutes post dose corresponding to ICE class III The combination of the three endpoints for the positive control, 5% Benzalconium chloride was 3 x IV. Therefore, the positive controle is classified as "Corrosive/Severe irritant" as expected. The combination of the three endpoints for the negative control, physiological saline was 3 x I. Therefore, the positive controle is classified as "No category" as expected. The results obtained, under this experimental conditions enable to conclude that the test item Cetonal does not have to be classified in category 1 "irreversible effects on eye" according to the CLP Regulation (EC) No. 1272/2008.