Reaction mass of 3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate and 3,7-dimethyloctyl 3-methyl-2-butenoate and citronellyl 3-methylcrotonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 07, 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:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Citronellyl 3-methylcrotonate

EC Number:

244-019-4

EC Name:

Citronellyl 3-methylcrotonate

Cas Number:

20770-40-5

Molecular formula:

C15H26O2

IUPAC Name:

3,7-dimethyloct-6-en-1-yl 3-methylbut-2-enoate

Constituent 2

Reference substance name:

3,7-dimethyloctyl 3-methyl-2-butenoate

EC Number:

275-360-7

EC Name:

3,7-dimethyloctyl 3-methyl-2-butenoate

Cas Number:

71383-07-8

Molecular formula:

C15H28O2

IUPAC Name:

3,7-dimethyloctyl 3-methylbut-2-enoate

Constituent 3

Reference substance name:

3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate

Cas Number:

74499-48-2

Molecular formula:

C15H26O2

IUPAC Name:

3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

Identity: SINODOR CQ U/A - 6099932
Batch-No.: VE00172025
Purity: > 97% (sum isomers)
Stability in Solvent: Not relevant
Storage: At room temperature, protected from light and moisture
Expiration Date: May 2013

Test animals / tissue source

Species:

other: Bovine eyes

Strain:

other: Corneas

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the
abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were
transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at
ambient temperature. The corneae were isolated on the same day after delivery of the eyes
and used directly for the BCOP test.
All eyes were carefully examined macroscopically for defects. Those presenting defects such
as vascularization, pigmentation, opacity and scratches were discarded. The cornea was
carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of
tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in
the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and
checked finally with a view box for defects listed above.
Each isolated cornea was mounted in a specially designed cornea holder according to the
description given in OECD guideline 437, annex III, that consists of anterior and posterior
compartments, which interface with the epithelial and endothelial sides of the cornea,
respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring)
of the posterior part of the holder. The cornea was gently flattened over the O-ring but
stretching was avoided. After the anterior part of the holder was positioned on top of the
cornea and fixed in place with screws, both compartments of the holder were filled with
complete medium. The posterior compartment was filled first to return the cornea to its
natural convex position. Care was taken to assure no air bubbles were present within the
compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for one hour
at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

The anterior compartment received the test item or negative or positive control at a volume
of 0.75 mL on the surface of the corneae.
The test item was tested undiluted. The positive control 2-Ethoxyethanol was tested neat.
Saline was used as negative control item.

Duration of treatment / exposure:

10 minutes
120 minutes

Number of animals or in vitro replicates:

Negative control: 3 Corneae
Positive control: 3 Corneae
Test item: 3 Corneae

Details on study design:

The experiment was performed to determine an irritation effect of the test item on the
corneal opacity.
The anterior compartment received the test item or negative or positive control at a volume
of 0.75 mL on the surface of the corneae and was incubated at 32 ± 1 °C in the water-bath,
while the corneae were in a horizontal position.
The test item was tested undiluted. The positive control 2-Ethoxyethanol was tested neat.
Saline was used as negative control item.
The incubation time lasted ten minutes.
After the test item or control items, respectively, were rinsed off from the application side
with saline, fresh cMEM was added into the anterior compartment. The corneae were then
incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a 2nd opacity
reading (t130).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Nafluorescein
solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was
placed in the anterior compartment. Corneae were incubated again in a horizontal position
for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot
of the mixed complete medium from the posterior chamber was measured
spectrophotometrically at 490 nm (OD490).

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae,
and displays a numerical opacity value. This value was recorded in a table. The
opacitometer was calibrated as described in the manual and the opacity of each of the
corneae was determined by reading each holder placed in the photoreceptor compartment
for treated cornea.
The basal opacity of all corneae was recorded. Each corneae with a value of the basal
opacity > 7 was discarded. Sets of three corneae were used for treatment with the test items
and the negative and positive controls.
Complete medium was completely removed from the anterior compartment and replaced by
the test item, positive or negative control. The anterior compartment was plugged. The
cornea was turned to a horizontal position and slightly rotated to ensure uniform covering of
the cornea with the test item and was incubated in a water-bath at 32 ± 1 °C for ten minutes
followed by two hours incubation after replacing the test item solution by cMEM in a vertical
position. Afterwards, the opacity value was determined again.

Permeability Determination
Following the opacity readings, the permeability endpoint was measured as an indication of
the integrity of the epithelial cell sheets. After the final opacity measurement was performed,
the complete medium was removed from the anterior compartment and replaced by 1 mL of
a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a
horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the
posterior compartment was removed, well mixed and the optical density at 490 nm (OD490)
was determined with a spectrophotometer.

Criteria for Determination of a Valid Test
The test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the
in vitro irritation score of the negative control was ≤ 3.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

3.01

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corneal Opacity :
The opacity reading for the test article was 2.67, 1.67 and 4.67.
The mean opacity reading for the negative control was 1.33.
The opacity reading for the positive control was 78.67, 74.67 and 81.67.

Corneal Permeability:
The optical density for the test article was 0.004, 0.001 and -0.003.
The mean optical density for the negative control was 0.048.
The optical density for the positive control was 0.486, 0.209 and 0.318.

Applicant's summary and conclusion

Interpretation of results:

not classified

Conclusions:

With the negative control (saline) neither an increase of opacity nor permeability of the
corneae could be observed (mean in vitro irritation score 2.05).
The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of
the corneae (mean in vitro irritation score 83.40) corresponding to a classification as
corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item SINODOR CQ U/A - 6099932 caused a very
slight increase of the corneal opacity, whereas a rise of the permeability values was not
observed. The calculated mean in vitro irritation score was 3.01 (threshold for corrosivity /
severe irritancy: ≥ 55.1). According to OECD 437 the test item is classified as not corrosive /
not severe irritant to the eye.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of

SINODOR CQ U/A - 6099932 by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item

SINODOR CQ U/A - 6099932, the positive, and the negative controls were applied to

corneae and incubated for 10 minutes at 32 ± 1 °C. The posterior chamber contained MEM

medium supplemented with sodium bicarbonate and L-glutamine and 1% fetal calf serum

(FCS) (complete medium = cMEM). After the incubation phase the test item, the positive,

and the negative controls were each rinsed from the corneae. Further, the corneae were

incubated for another 120 minutes at 32 ± 1 °C in complete medium, and opacity was

measured a second time (t130).

After the opacity measurements permeability of the corneae was determined by measuring

spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal

position for 90 minutes at 32 ± 1 °C.

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase

of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of

the corneae corresponding to a classification as corrosive / severe irritant to the eye

(CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item SINODOR CQ U/A - 6099932 caused a very

slight increase of the corneal opacity. Permeability effects were not observed. The calculated

mean in vitro irritation score was 3.01. According to OECD 437 the test item is classified as

not corrosive / not severe irritant to the eye.

In conclusion, according to the current study and under the experimental conditions

reported, the test item SINODOR CQ U/A - 6099932 is not corrosive / not severe irritant to

the eye (CLP/EPA/GHS (Cat 1)).