Hydrocarbon waxes (petroleum), oxidized, Me esters

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2017 to 06 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Confidential

IUPAC Name:

Confidential

Test material form:

solid

Details on test material:

- Appearance / physical state: Dark brown paste
- Storage conditions: Room temperature in the dark

Test animals / tissue source

Species:

human

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg of test material

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

Two

Details on study design:

PURPOSE OF STUDY
- Eye irritation is generally defined as "the production of reversible changes in the eye". The potential for chemical induced eye irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. It is usually determined in vivo in the Draize rabbit eye irritation test as described in OECD guideline 405. In a pre-validation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the EpiOcular human cornea model and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.
- The EpiOcular Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
- The test consists of a topical exposure of a human reconstructed cornea model to the neat test item followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

DOSE SELECTION
- Approximately 50 mg of the test item (83.3 mg/cm2 according to guideline) was tested topically on duplicate EpiOcular tissues.
- The tissue was placed back into the culture medium after dosing and incubated at standard culture conditions for 6 hours.

EPIOCULAR KIT COMPONENTS
- Lot numbers: 23764 and 23774
- Sealed 24-well plate: Contains 12/24 inserts with EpiOcular tissues on agarose
- Serum-free test medium: DMEM-Medium
- Positive control: Methyl Acetate (CAS#79-20-9)
- 12-well plate: Holding plate
- 24-well plates: For MTT viability assay
- 6-well plates: For storing inserts, or for topically applying test agents
- Ca++Mg++-Free D-PBS: Dulbecco's Phosphate Buffered Saline

MTT-100 ASSAY KIT COMPONENTS
- 1 vial (2 mL): MTT concentrate
- 1 vial (8 mL): MTT diluent (supplemented DMEM) for diluting MTT concentrate prior to use in the MTT assay
- 1 bottle (60 mL): Extractant solution (lsopropanol) for extraction of formazan crystals

CELL CULTURE
- EpiOcular kits and MTT-100 kits were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organised basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular tissues (surface 0.6 cm2) were cultured on specially prepared cell culture inserts (MILLICELL , 10 mm Ø).
- EpiOcular tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate on Tuesday. On day of receipt of the EpiOcular tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
- Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70 % isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the assay medium. After one hour, the assay medium was replaced by 1 mL of fresh assay medium at 37 °C and the EpiOcular tissues was incubated at standard culture conditions overnight (19 or nearly 17 hours, respectively).

MTT SOLUTION
- On the day of the experiment a MTT solution of 1 mg/mL in DMEM was prepared.

ASSESSMENT OF DIRECT MTT REDUCTION BY THE TEST ITEM
- Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose, approximately 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
- Since the test item was shown to reduce MTT, an additional test using freeze-killed tissue controls (killed controls = KC) was performed to determine a correction factor for calculating the true viability in the main experiment.

ASSESSMENT OF COLOURED OR STAINING MATERIALS
- Coloured test items or test items which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colorant bound to the tissue and would be extracted together with MTT. Therefore, each test item had to be checked for its colourant properties.
- For this purpose, each approximately 50 mg of the test item was added to 1.0 mL of water and to 2 mL isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 in air for one hour, the isopropanol mixture for 2 to 3 hours at room temperature.
- Since the test item only dyed water and isopropanol slightly yellow, it was not considered as possibly interacting with the MTT measurement. It was therefore unnecessary to measure the absorbance at a wavelength at 570 nm and an additional test with viable tissues (with medium instead of MTT addition) did not have to be performed.

EXPERIMENTAL PROCEDURE
- After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
- After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 mg (test item) or 50 μL (controls) topically on the EpiOcular tissues.
- The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 % RH) for 6 hours.
- At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature).
- Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. The test item utilised a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts are mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed assay medium (room temperature) in a pre-labelled 12-well plate for 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
- At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5 % CO2 (post-treatment incubation).

MTT ASSAY
- At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
- Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were in the first experiment extracted overnight (about 20 hours) at 2-8 °C in the dark and in the second experiment immediately extracted (shaken for about 2 hours at room temperature). The tissues were not pierced. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically without piercing. For this procedure, it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
- The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s).
- The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

KILLED CONTROLS FOR ASSESSMENT OF RESIDUAL TEST ITEM REDUCTION OF MTT
- Since the test item was shown to reduce MTT, only test item that remained bound to the tissue after rinsing, resulting in a false MTT reduction signal, presented a problem. To demonstrate that residual test item was not acting to directly reduce the MTT, a functional check on freeze-killed tissues (KC) was performed in the definitive assay to show that the test material is not binding to the tissue and leading to a false MTT reduction signal.
- Freeze killed tissues are prepared by placing untreated EpiOcular constructs (in a 24-well plate) in the -20 °C freezer overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles). Once frozen, the tissue may be stored indefinitely in the freezer.
- Two killed tissues were treated with the test item in the normal fashion. All assay procedures were performed as for the viable tissue. Two freeze-killed tissues treated with sterile deionised water (negative control) were tested in parallel since a small amount of MTT reduction was expected from the residual NADH and associated enzymes within the killed tissue.

DATA RECORDING
- The data generated were recorded in the laboratory protocol. The results were presented in tabular form, including experimental groups with the test item and the controls.

DATA EVAUATION
- The mean OD value of the blank control wells (OD Blk) for each experiment was calculated.
- The mean value of the two aliquots for each tissue was calculated.
- The mean OD Blk from each mean OD value of the same experiment was subtracted (blank corrected values).
- The mean value of the two relating tissues for each control (negative control (NC) and positive control (PC) and test item (TI) was calculated (ODTI, ODNC, ODPC).
- The mean OD value of the negative control corresponds to 100% viability.
- The Corrected negative control OD = Negative Control OD – OD Blk = 100% Viability

CALCULATIONS FOR VIABILITY TESTS ONLY
- The percentage viability of each of the two relating tissues was calculated relative to the negative control (100 % control) for each control and test item using the equation viability [%] = 100 * [(OD TI / OD PC) / ODNC] / mean OD NC
- The difference in viability between duplicate tissues was calculated. The test is considered invalid if the difference is > 20 %.
- The mean test item viability (TI viability) was calculated and the test item was classified according to the prediction model.

CALCULATIONS FOR VIABILITY PLUS KILLED CONTROL (KC) TESTS
- The OD values of the freeze-killed tissue experiment (each two tissues and two replicates) were determined and blank corrected The mean value of the two replicates for the negative control (NC) and test item (TI) was calculated (test item: ODTI_KC; negative control: ODNC_KC).
- The mean ODNC_KC was subtracted from mean ODTI_KC to determine the individual freeze-killed tissue values for the test item (ODKC). The equation used was ODKC = ODTI_KC – meanODNC_KC. The ODKC control represents the amount of reduced MTT due to direct reduction by test item residues.
- The viability of the two relating tissues was calculated according to the equation TestItemKCViability [%] = 100 * ODKC / meanODNC.
- The difference in viability between duplicate tissues was calculated. The test is considered invalid if the difference is > 20 %.
- The mean test itemKC viability was calculated and was subtracted from the test item viability (TI viability) to determine the KC corrected test item viability. The equation used was KC corrected test item viability = TI viability – mean TestItemKC Viability.
- The test item was classified regarding the freeze-killed tissue corrected viability according to the prediction model.

PREDICTION MODEL
- If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant.
- If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.
- According to OECD guideline 492, a single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60 ± 5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

ACCEPTABILITY OF ASSAY
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5.
2) The mean relative viability of the positive control is below 50 % of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: percentage cell viability

Remarks:

true viability

Run / experiment:

Experiment 1

Value:

58.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability reported as 59.1 % before correction factor taken into account

Results 2

Irritation parameter:

other: percentage cell viability

Remarks:

true viability

Run / experiment:

Experiment 2

Value:

30.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: cell viability reported as 30.5 % before correction factor taken into account

Other effects / acceptance of results:

RESULTS
- Results after treatment for 6 hours with test item and the controls are shown in the attached table.
- Historical data are shown in Annex 1 (attached).
- The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol only led to a weak change in colour (slightly yellow). Therefore, an additional test with viable tissues without MTT addition was not necessary.
- Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent showed blue colour. Therefore, an additional test with freeze-killed tissues was performed.
- The corrected mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 58.8% in the 1st experiment (threshold for irritancy: ≤ 60%). Since the result was borderline, the test was performed for a second time according to the recommendation of the OECD 492 guideline and the Sponsor’s request. In the second experiment, a corrected cell viability of 30.2% was determined, consequently the test item was classified as irritant to eye.

ACCEPTANCE CRITERIA
- The negative control OD was > 0.8 and < 2.5 (values between 1.703 and 1.876).
- The mean relative viability of the positive control was below 50 % of the negative control viability (17.8 % and 42.6 %).
- The difference of viability between the two relating tissues of a single item was < 20% (values between 0.0% and 11.1%) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the killed controls (items and negative control) which were calculated as percent values related to the viability of the relating negative control.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the experimental conditions reported, the test item possesses eye irritating potential.

Executive summary:

GUIDELINE

An in vitro study was conducted to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. The investigation was performed in compliance with the OECD Guideline for Testing of Chemicals 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July 2015) and the MatTek Corporation Protocol: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular Model (29 June 2015).

METHODS

The test item proved to be an MTT reducer in the MTT pre-test. Its intrinsic colour was not intensive, and it dyed water and isopropanol in the colour interference pre-test only slight yellowish. Therefore, an additional test with freeze-killed tissues to determine a correction factor for calculating the true viability in the main experiment was performed, but the additional test with viable tissues (without MTT addition) was not necessary. Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 μL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues. 

 

RESULTS

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50 % compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20 % in the same run (for test item tissues, positive and negative control tissues). Irritating effects were observed following incubation with test item in both experiments. Compared with the value of the negative control the corrected relative mean absorption values corresponding to the viability of the tissues decreased below 60 % (58.8 % and 30.2 %).

 

CONCLUSION

Under the experimental conditions reported, the test item possesses eye irritating potential.