3,4,5-trimethoxycinnamic acid

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 26, 2013

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
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.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing:
Bovine eyes were used as soon as possible after slaughter
- indication of any existing defects or lesions in ocular tissue samples:
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.
- Indication of any antibiotics used: no

Vehicle:

unchanged (no vehicle)

Remarks:

Since no workable suspension of 3,4,5-TRIMETHOXYCINNAMIC ACID in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
3,4,5-TRIMETHOXYCINNAMIC ACID was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (315.6 to 384.3 mg).
- Concentration (if solution):
The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea.

VEHICLE
No vehicle used

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240  10 minutes at 32  1C.

Number of animals or in vitro replicates:

3 replicates per treatment (negative control, positive control and test item)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
3 replicates per treatment (negative control, positive control and test item)

NEGATIVE CONTROL USED
A negative control, physiological saline was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED
20% (w/v) Imidazole (Merck Schuchardt OHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. 3,4,5-TRIMETHOXYCINNAMIC ACID was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (315.6 to 384.3 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240  10 minutes at 32  1C.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
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.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90  5 minutes at 32  1C.
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 l of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Irritation parameter:

in vitro irritation score

Value:

20

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Value:

19

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Value:

0.111

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The individual in vitro irritancy scores for the negative controls ranged from 0.1 to 1.6.
- Acceptance criteria met for positive control:
The individual positive control in vitro irritancy scores ranged from 110 to 135 .
- Range of historical values if different from the ones specified in the test guideline: NA

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since 3,4,5-TRIMETHOXYCINNAMIC ACID induced an IVIS between 3 and 55, no prediction on the classification can be made.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 3,4,5-TRIMETHOXYCINNAMIC ACID as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of 3,4,5-TRIMETHOXYCINNAMIC ACID was tested through topical application for approximately 240 minutes. 

The study procedures described in this report were based on the most recent OECD guideline (OECD 437, adopted July 26, 2013).

Batch CH03216 / E1A of 3,4,5-TRIMETHOXYCINNAMIC ACID was a light yellow crystallinesolid. Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (20% (w/v) Imidazole) was 123 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

3,4,5-TRIMETHOXYCINNAMIC ACID induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 20 after 4 hours of treatment.

In conclusion, since 3,4,5-TRIMETHOXYCINNAMIC ACID induced an IVIS between 3 and 55, no prediction on the classification can be made. 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 28 July 2015

Deviations:

yes

Remarks:

The deviation was assessed to not influence the outcome of the test

GLP compliance:

yes

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
EpiOcularTM (OCL-200-EIT MatTek Corporation, Validated in Test Facility Study Number 509657, Lot: 23497 kit C)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
No correction was made for the purity/composition of the test item.
The solid test item (55.0 to 55.2 mg) was applied directly on top of the skin tissue.
Any residual volumes were discarded.

Duration of treatment / exposure:

6 hours ± 15 minutes at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

- RhCE tissue construct used, including batch number
EpiOcularTM (OCL-200-EIT MatTek Corporation, Validated in Test Facility Study Number 509657, Lot: 23497 kit C)

- Doses of test chemical and control substances used
The solid test item (55.0 to 55.2 mg) was applied directly on top of the skin tissue. Two tissues were treated with 50 μl Milli-Q water (negative control) and 2 tissues with 50 μl Methyl Acetate (positive control) respectively.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable):
2

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader (linearity range 0.092 – 2.185).

- Description of the method used to quantify MTT formazan
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 ml MTT-medium
(1.0 mg/ml). The tissues were incubated for 180 ± 10 minutes at 37°C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 ml isopropanol for 2 - 3 hours at room temperature with gentle shaking 2 hours in the dark.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader (linearity range 0.092 – 2.185).
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

- Acceptable variability between tissue replicates for positive and negative controls
The mean relative tissue viability of the positive control should be <50% relative to the negative control.

- Acceptable variability between tissue replicates for the test chemical
The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
The difference between the % tissue viabilities of the two identically treated replicates should be <20%.

Irritation parameter:

other: Mean tissue viability

Value:

22

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: SD = 2.5%

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
3,4,5-TRIMETHOXYCINNAMIC ACID was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint.
The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.003 and 0.004, respectively. Therefore it was concluded that the test item did not induce color interference.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 22%. Since the mean relative tissue viability for the test item was below 60% it is considered to be potentially irritant or corrosive to the eye.
The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 30%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In conclusion, 3,4,5-TRIMETHOXYCINNAMIC ACID is potentially irritant or corrosive in the EpiOcularTM test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 3,4,5-TRIMETHOXYCINNAMIC ACID. For this purpose the test item was topicallyapplied on the Reconstructed Human EpiOcularTM Model.

The possible eye hazard potential of the test item was tested through topical application for 6 hours.

The study procedures described in this report were based on the most recent OECD guideline (OECD 492, Adopted 28 July 2015).

Batch CH03216 / E1A of the test item was a light yellow crystalline solid with a purity of approximately 99%. The test item (55.0 to 55.2 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes.

After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 30% after 6 hours ± 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 22%. Since the mean relative tissue viability for the test item was below 60% after 6 hours ± 15 minutes treatment no prediction about the category can be made.

In conclusion, the test item is potentiallyirritant or corrosive in the EpiOcularTM test underthe experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Two in-vitro studies are available on the potential for eye irritation of 3,4,5 -Trimethoxycinnamic acid. The BCOP study concluded that no conclusion can be made on the CLP classification. The RhCE study (EpiOcular) concluded that

3,4,5 -Trimethoxycinnamic acid should be classified as either Category 2 (irritating) or Category 1 (irreversible damage). Because the BCOP study did not show indications of irreversible damage but did indicate irritation of the eye and the EpiOcular test similarly found indications of eye irritation, 3,4,5 -Trimethoxycinnamic acid was classified as Category 2: (irritating).