Reaction mass of rel-{(1R,2S)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol and rel-{(1S,2R)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (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:

[(1R,2S)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl]methanol; [(1R,2S)-1-methyl-2-[(2S)-5-methylhex-4-en-2-yl]cyclopropyl]methanol; [(1S,2R)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl]methanol; [(1S,2R)-1-methyl-2-[(2S)-5-methylhex-4-en-2-yl]cyclopropyl]methanol

EC Number:

942-597-9

Cas Number:

1655500-83-6

Molecular formula:

C12H22O

IUPAC Name:

[(1R,2S)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl]methanol; [(1R,2S)-1-methyl-2-[(2S)-5-methylhex-4-en-2-yl]cyclopropyl]methanol; [(1S,2R)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl]methanol; [(1S,2R)-1-methyl-2-[(2S)-5-methylhex-4-en-2-yl]cyclopropyl]methanol

Test material form:

other: Liquid

Details on test material:

Batch VE00340479
Purity 87.8 % (Sum of the four isomers)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Test animals / tissue source

Species:

other: Bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Bovine eyes were used as soon as possible after slaughter on the same day.
The eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

The test substance was tested neat

Controls:

other: not applicable, in vitro test (see report)

Amount / concentration applied:

750 μl of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or test substance was introduced onto the epithelium of thecornea.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

Observation period (in vivo):

not applicable, in vitro test (see report)

Duration of post- treatment incubation (in vitro):

After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium

Number of animals or in vitro replicates:

not applicable, in vitro test (see report)

Details on study design:

The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean after 10 mins of treatment

Value:

ca. 14.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

No prediction on the classification can be made

Other effects / acceptance of results:

Rosyfolia induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of IVIS of 14.1 after 10 minutes of treatment.


IVIS UN GHS
< 3 No Category
> 3; < 55 No prediction can be made
> 55 Category 1



Since rosyfolia induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.


Acceptance criteria:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within the laboratory historical mean value
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range

All acceptance criteria were met.

Any other information on results incl. tables

The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

Table 1 Summary of opacity, permeability and in vitro scores
Treatment		Mean Opacity1	Mean Permeability1	Mean In vitro Irritation Score1, 2
Negative control		-1	0	-0.7
Positive control (Benzalkonium Chloride)		91	3.571	144.2
Rosyfolia		13	0.051	14.1
1 Calculated using the negative control mean opacity and mean permeability values.
2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

The treated corneas showed opacity values ranging from 12 to 15 and permeability values ranging from 0.033 to 0.067. The corneas were slightly turbid with spots after the 10 minutes of treatment with rosyfolia. No pH effect of the test substance was observed on the rinsing medium.

Rosyfolia induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 14.1 after 10 minutes of treatment.

Since rosyfolia induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Applicant's summary and conclusion

Interpretation of results:

other: Rosyfolia mean in vitro irritancy score of 14.1 showed a lack of severe irritation after the exposure period

Remarks:

GHS Category 2

Conclusions:

Rosyfolia induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 14.1 after 10 minutes of treatment. Since rosyfolia induced an IVIS > 3 ≤ 55, no prediction on the classification can be made

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. The methods and procedures used in this assay were consistent with OECD Test Guideline 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2013) and EC Guideline B.47 under GLP. Rosyfolia was topically applied undiluted to the epithelium of the bovine cornea for 10 minutes. After exposure, the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein. A negative control of saline and a positive control of 10% benzalkonium chloride were included concurrently in the experiments. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 144 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Rosyfolia induced ocular irritation through both endpoints, with an IVIS > 3 ≤ 55, indicating that no prediction on the classification can be made. However, the

resulting mean in vitro irritancy score of 14.1 after 10 minutes of treatment does indicate that rosyfolia is not a severe irritant.