Hexadecyl hydrogen maleate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From May 6th, 2019 to May 9th, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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 June 25th, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hexadecyl hydrogen maleate

EC Number:

220-942-8

EC Name:

Hexadecyl hydrogen maleate

Cas Number:

2944-06-1

Molecular formula:

C20H36O4

IUPAC Name:

(2Z)-4-(Hexadecyloxy)-4-oxo-2-butenoic acid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

The commercially available EpiOcularTM kit was used. The EpiOcularTM tissues were procured from MatTek (In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia) (Designation of the kit: OCL-200-EIT; Day of delivery: May 7th, 2019; Batch no.: 30605). The EpiOcularTM tissues consist of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. EpiOcularTM tissues consist of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

52.1 mg of test material on 0.6 cm2 of tissue 1
52.0 mg of test material on 0.6 cm2 of tissue 2

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours and 15 minutes

Number of animals or in vitro replicates:

2 in vitro replicates

Details on study design:

CHEMICALS AND MEDIA:
- MTT solution: MTT solution (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), which can be reduced to a blue formazan, was prepared by the testing facility. A MTT stock solution of 5 mg/ml in DPBS buffer was prepared and stored in aliquots of 2 ml at – 20 ± 5 °C. 2 ml of the stock solution were thawed and diluted with 8 mL of assay medium (resulting in 1 mg/ml). This MTT-solution with the concentration of 1 mg/mL was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution was thawed and diluted with assay medium directly before use.
- DPBS-Buffer: “Dulbecco`s Phosphate Buffered Saline” (DPBS) was used for the rinsing of the tissues. A subset was procured; the other subset was prepared by the testing facility. Composition of the subset procureed: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 L. Composition of the subset prepared by the testing facility: KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 2H2O 1.44 g, H2O ad 1 L. The buffer procured was used for rinsing the test item from the tissues. The buffer prepared by the testing facility was only used for preparing the MTT concentrate.
- MEM with Phenol Red for Pre-Test: Serum-free MEM (Minimum Essential Medium)
- Assay Medium for Main Test: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium)

PRE-TESTS
The test item was ground to a fine powder before usage.
- Assessment of Direct Reduction of MTT by the Test Item
The test item was tested for the ability of direct MTT reduction. The test item was tested for the ability of direct MTT reduction. 51.3 mg of the solid test item were added to 1 ml of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and ≥ 95% relative humidity for 3 hours. 1 ml of MTT solution plus 50 µl of H2O demin. was used as negative control. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
- Assessment of Coloured or Staining Test Items
52.2 mg of the test item were added to 2 ml isopropanol, incubated in 6-well plates on an orbital shaker for 2 hours at room temperature. Then, two 200 µl aliquots of the resulting solution and two 200 µl aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm. After subtraction of the mean OD for isopropanol, the mean OD of the test item solution was 0.073 (≤ 0.08). Therefore, the main test was performed without colorant controls.

MAIN TEST
- On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. The solid test item was ground. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 ml fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 16 hours 29 minutes.
- After overnight incubation, the tissues were pre-wetted with 20 µl Ca2+/Mg2+-free DPBS buffer and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 29 minutes. After that, 50 µl of the controls and a defined amount of the test item were applied in duplicate in one- minute- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
- At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with Ca2+/Mg2+-free DPBS. Then, the tissues were immediately transferred into 5 ml of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was removed from the medium, the medium was decanted and the inserts were blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 ml assay medium. For post-treatment incubation, the tissues were incubated for 18 hours 15 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity. After the post-treatment incubation, the MTT assay was performed.

MTT ASSAY AND EXTRACTION
- A 24-well-plate was prepared with 300 µl freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 190 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
- At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 ml isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
- The inserts were removed from the 6-well plate and discarded (without piercing the tissues). The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 µl solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µl isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Value:

2.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the EpiOcularTM test at LAUS GmbH was demonstrated in a proficiency study. For this purpose, 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. OD of negative control: 1.7
- Acceptance criteria met for positive control: Yes. % tissue viability of positive control: 31.8%
- Acceptance criteria met for variation within replicates: Yes, 0.7% variation within replicates for negative control, 8.9% variation within replicates for positive control, 0.2% variation within replicates for test item.
- Range of historical values: negative and postive controls are in the range of historical values.

Applicant's summary and conclusion

Interpretation of results:

other: According to OECD 492, the test does not allow discrimination between eye irritation/reversible effects (Cat 2) and serious eye damage/irreversible effects on the eye (Cat1). For this reason further testing with other suitable test methods is required.

Conclusions:

Under the conditions of the test, the substance is considered either eye irritant or inducing serious eye damage

Executive summary:

Determination of Eye Hazard Potential of the test substance using the EpiOcularTM Reconstructed human Cornea-like Epithelium (RhCE) test method was performed following OECD 492. The test itemwas applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. One valid experiment was performed. After treatment and rinsing of the test item from the tissue, the cell viability of the tissues was evaluated by MTT test for cell viability. Demineralised water was used as negative control and methyl acetate was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion. The positive control showed clear eye irritating effects. The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 2.5 %, which is below the threshold discriminating for eye irritation potential (≤ 60%).

Therefore, under the conditions of the test, Hexadecyl hydrogen maleate is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test. However, according to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required.