2-methyl-3-(p-tolyl)propionaldehyde

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 03, 2021 to August 02, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD 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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-3-(p-tolyl)propionaldehyde

EC Number:

255-410-4

EC Name:

2-methyl-3-(p-tolyl)propionaldehyde

Cas Number:

41496-43-9

Molecular formula:

C11H14O

IUPAC Name:

2-methyl-3-(4-methylphenyl)propanal

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing". 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.

Source: Bovine eyes from young cattle were obtained from the abattoir (Mahan Packing Company Inc., Bristolville, OH), where the eyes were excised by an abattoir employee as soon as possible after slaughter.

Transport: Eyes were collected and transported in Hank's balanced salt solution (HBSS) supplemented with antibiotics and antimycotics in a suitable container under cooled conditions.

Identification: Each cornea was mounted in a corneal holder. The posterior and anterior halves of the cornea holder are each engraved by the manufacturer with a one-digit or two-digit holder number. Posterior and anterior halves of the holder had matching holder numbers. A holder with a unique number was used to mount each cornea, and the cornea remained mounted in the same holder for the duration of the study.

Preparation of corneas: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the HBSS and holding them in the light for gross examination. Those exhibiting defects were discarded.
Corneas were isolated by making an incision through the sclera with a scalpel and cutting a circumference around the cornea with dissecting scissors. At least 2 mm of sclera was left around the cornea circumference to aid in handling, and the iris and lens were removed. The isolated corneas were stored in a dish with cMEM (complete Minimum Essential Medium (Gibco, Gaithersburg, MD) containing 2% (v/v) L-glutamine (Gibco, Gaithersburg, MD) and 1% (v/v) Fetal Bovine Serum (Gibco, Gaithersburg, MD)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany). The posterior and anterior halves of the cornea holder were each engraved by the manufacturer with a one-digit or two-digit holder number. Posterior and anterior halves of the holder had matching holder numbers. A holder with a unique number was used to mount each cornea. The cornea remained secured in the holder for the duration of the study and was identified by the unique holder number. Each cornea was mounted with the endothelial side against the 0-ring of the posterior half of the holder. The anterior portion of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM at 32 ± 1°C. The corneas were incubated for 60 minutes and 11 seconds at 32 ± 1°C.

Cornea selection and opacity reading: After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (ElectroDesign OP-KIT, France). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. No corneas had an initial opacity reading higher than 4. Three corneas were selected at random for each treatment group.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of the test article was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for between 9 minutes and 57 seconds and 10 minutes and 11 seconds at 32 ± 2°C.

Duration of post- treatment incubation (in vitro):

The corneas were incubated for between 1 hour and 44 minutes and 59 seconds to 2 hours and 32 seconds at 32 ± 2°C

Number of animals or in vitro replicates:

Negative control: 3 corneas
Positive control: 3 corneas
Test item: 3 corneas

Details on study design:

- Test Article Preparation
The test article was tested neat (undiluted) as formulated by the Sponsor, therefore no correction for the purity/composition of the test article was performed.

- Treatment of Corneas and Opacity Measurements
For each treatment group, the medium from the anterior compartment was removed and 750 µL of the negative control (0.9% NaC1) or positive control (ethanol) or test article was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position for between 9 minutes and 57 seconds and 10 minutes and 11 seconds at 32 ± 2°C. After the incubation, the test and control articles were rinsed three times with cMEM with the chamber closed. No color change was observed in the phenol red indicator in the wash solution of the control articles. The test article produced a slight purple color from the phenol red indicator in the first wash only, indicating a basic pH change.
For all treatment groups, following washing with phenol-red containing cMEM, the anterior compartment was washed once with phenol red free cMEM and then refilled with fresh phenol red free cMEM. Subsequently the corneas were incubated for between 1 hour and 44 minutes and 59 seconds to 2 hours and 32 seconds at 32 ± 2°C. After the completion of the incubation period each cornea was inspected visually. No spots or irregularities were observed in the negative control nor test article treatment group corneas. All three corneas in the positive control group were opaque.

- Opacity Measurement
The opacity of a cornea was measured by the diminution of light passing through the cornea.
The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = IT - Io
With IT the measured illuminance of the cornea after treatment, and /0 the measured illuminance of the cornea prior to treatment.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

-Application of Sodium Fluorescein
Following the final opacity measurement, permeability of the cornea to sodium (Na) fluorescein (SIGMA, St. Louis, MO) was evaluated. Media from both chambers was removed. The posterior chamber was filled with fresh cMEM without phenol red. The anterior chamber was filled with 1 mL of 4 mg Na-fluorescein/mL cMEM solution.
Following application of Na-fluorescein, the holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the Na-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 91 minutes and 51 seconds at 32 ± 2°C.

- Permeability Determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The medium from the positive control corneas was diluted 1 volume : 6 volumes of phenol-red free cMEM to bring the optical density into the acceptable range (< 1.5000 AU). The optical density at 490 nm (01:0490) of each sampling tube was measured in triplicate using a microplate reader (Biotek Cytation 5; Winooski, VT) and reported in Absorbance Units (AU).
The diluted solution from the positive control treated corneas produced acceptable OD490 values (<1.5000 AU) that were used in subsequent calculations. The mean 0D490 for each treatment was calculated using cMEM corrected OD490 values. The background was corrected by averaging nine wells containing cMEM media without sodium fluorescein.

-Cornea Fixation
Corneas were fixed according to standard methods". After the sodium fluorescein solution was removed from the posterior chamber of each cornea, the corneas were washed once (posterior and anterior chamber) with 1x PBS. Then, the cornea holders were disassembled, and each cornea transferred into a labelled culture plate filled with room temperature PBS.
The PBS was aspirated, and 2 mL of pure methanol was added to each cornea. Corneas were incubated in methanol for 27 minutes and 16 seconds at room temperature. The methanol was aspirated, and the corneas were washed with approximately 2 mL of 1x PBS per cornea, followed by aspiration. The corneas were stored in a fresh volume of 1x PBS at 2-8°C protected from light.

-Acceptability criteria:
The assay was considered acceptable because:
• The positive control gave an in vitro irritancy score that falls within two standard deviations of the current historical mean and was greater than the minimum IVIS observed in the historical controls.
• The negative control responses resulted in opacity and permeability values that were less than the upper limits of the laboratory historical range.

-Interpretation:
The mean opacity and mean permeability values (OD490) was used to calculate an in vitro irritation score (IVIS) for each cornea, with the IVIS scores averaged for each treatment group:
IVIS = mean opacity value + (15 x mean OD490 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test article induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test articles as inducing serious eye damage (UN GHS Category 1) and test articles not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
IVIS =< 3: no category under UN GHS
3 > IVIS =< 55: no prediction can be made under UN GHS
IVIS >55: Category 1 under UN GHS

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

2.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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. All results are within the acceptability range. The mean IVIS of the positive control (200 proof ethanol) was 45.3 and within two standard deviations of the current historical positive control mean, and greater than the minimum IVIS observed in the historical data for positive control (200 proof ethanol) (minimum observed mean IVIS 25.9). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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. All results are within the acceptability range. The mean IVIS of the positive control (200 proof ethanol) was 45.3 and within two standard deviations of the current historical positive control mean, and greater than the minimum IVIS observed in the historical data for positive control (200 proof ethanol) (minimum observed mean IVIS 25.9). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The average IVIS of 2.6 indicates that 2-methyl-3-(p-tolyl)propionaldehyde receives a UN GSH "No Category" classification for eye irritation or serious eye damage.

Executive summary:

The objective of this study is to determine the eye hazard potential of 2-methyl-3-(p-tolyl)propionaldehyde by the ability to induce opacity and increase permeability in an isolated bovine cornea. The method was based on the following OECD guideline:
• OECD 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, (adopted October 09, 2017).
This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of each test article was tested through topical application to isolated bovine corneas for approximately 10 minutes followed by approximately 2 hours of additional incubation and determination of opacity and permeability. Opacity was measured in lux and permeability measured in Absorbance Units (AU).
The experiment passed the acceptance criteria:

• The negative control responses for opacity (1.0 lux) and permeability (0.013 AU) were less than the upper limits of the laboratory historical range (maximum observed mean from historical runs, 1.7 lux, and 0.145 AU) indicating that the negative control did not induce irritancy on the corneas.
• The mean in vitro irritancy score (IVIS) of the positive control (200 proof ethanol) was 45.3 and within two standard deviations of the current historical positive control mean, and greater than the minimum IVIS observed in the historical positive control data (minimum observed mean IVIS 25.9).
It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
In Vitro Irritancy Scores are used to classify test articles according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations. Test articles producing an IVIS <3 result in a GHS UN designation of 'No Category'; Test articles producing an IVIS > 55, resulting in serious eye damage, receive a GHS UN designation of 'Category P. Test articles producing a score of 3 < IVIS < 55 result in a 'No prediction can be made' designation. 

2-methyl-3-(p-tolyl)propionaldehyde, was a clear to slightly yellow liquid. The test article was applied to the test system undiluted, according to OECD guidelines for formulated materials, for approximately 10 minute exposure and approximately 2 hour post-exposure incubation.
The test article induced a slight increase in opacity (mean 2.7 lux) and no impact on permeability (-0.003 AU), resulting in a mean IVIS of 2.6. In conclusion, the IVIS produced by 2-methyl-3-(p-tolyl)propionaldehyde in the BCOP test system results in a UN GHS designation of 'No Category' regarding eye irritation or serious eye damage.