6-O-palmitoylascorbic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD 492: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage (Adopted 28 July 2015) and under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2016

Materials and methods

Test guideline

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-O-palmitoylascorbic acid

EC Number:

205-305-4

EC Name:

6-O-palmitoylascorbic acid

Cas Number:

137-66-6

Molecular formula:

C22H38O7

IUPAC Name:

6-O-palmitoylascorbic acid

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

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.
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37 °C in 1.0 ml fresh prewarmed Assay Medium. Assay Medium was supplied by MatTek Corporation, Ashland MA, U.S.A.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

52.3 to 64.0 mg

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

two

Details on study design:

Before the assay was started the entire tissues was pre-wetted with 20 μL of Ca2+Mg2+-free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 µl MilliQ water (negative control) and 2 tissues with 50 µl Methyl Acetate (positive control) respectively. At least 50 mg solid (with a glass weight boat) were added into the 6-well plates on top of the tissues. After the exposure period with Ascorbyl palmitate (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. 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. Then, the tissues were transferred to a pre-labeled 6-well plate containing 2 ml isopropanol in each well and incubated for 2 - 3 hours at room temperature with gentle shaking or refrigerated for 18 ± 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. 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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

33

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Ascorbyl palmitate was checked for possible direct MTT reduction by adding the test item to MTT medium. Because a colour change was observed in the presence of MTT it was concluded that the test item interacted with the MTT endpoint. Ascorbyl palmitate was checked for colour interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.02 and 0.00, respectively. Therefore, it was concluded that the test item did not induce colour interference. Ascorbyl palmitate did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Hence, in addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Ascorbyl palmitate was 17% of the negative control tissues. The ODs of the test item treated viable tissues was corrected using he OD of the freeze-killed tissues.
The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 41%. The absolute mean OD570 of the negative control tissues was within 0.8 and 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Since the mean relative tissue viability for Ascorbyl palmitate was below 60% after 6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

Executive summary:

The possible eye hazard potential of Ascorbyl palmitate was tested through topical application on two tissues for 6 hours. A positive and a negative control were tested concurrently, each on two individual tissues.

The positive control had a mean cell viability of 41% after 6 hours ± 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within 0.8 and 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly.

Ascorbyl palmitate did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze-killed negative control treated tissue were used for cytotoxicity evaluation. The non-specific reduction of MTT by Ascorbyl palmitate was 17% of the negative control tissues. The ODs of the test item treated viable tissues was corrected using the OD of the freeze-killed tissues.

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 Ascorbyl palmitate compared to the negative control tissues was 33%. Since the mean relative tissue viability for Ascorbyl palmitate was below 60% after 6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

Finally, it is concluded that this test is valid and that Ascorbyl palmitate is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report.