Cyperus scariosus, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Jan 2018 to 2 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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

Test material

Test material information

Constituent 1

Reference substance name:

Cyperus scariosus, ext.

EC Number:

294-955-2

EC Name:

Cyperus scariosus, ext.

Cas Number:

91771-62-9

IUPAC Name:

Cyperus scariosus, ext.

Test material form:

liquid

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 ul

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Two

Details on study design:

8.4 Preparation of EpiOcular Tissues for Treatment
On the day of tissue receipt (16 January 2018) the tissues in its 24 well plate shipping container, was equilibrated to room temperature for about 15 minutes. In a 6 well plate 1 mL of assay medium was added to each well and warmed to approximately 37ºC in a Co2 incubator.
The 24 well plate shipping container was removed from the plastic bag under sterile conditions and surface disinfected by wiping with 70% ethanol. Each tissue in the 24 well plate was inspected for air bubbles between the insert and the agarose gel.
Tissues free of defects were selected and removed carefully from the 24 well plate using sterile forceps, agarose adhering to insert was removed gently by blotting on to sterile filter paper and placed in the 6 well plate containing 1 mL of assay medium, incubated at 37°C, 5% CO2 for 1 hour.
After 1 hour, assay medium was replaced with fresh assay medium and incubated further at 37°C, 5% CO2 for 16 hours.
8.5 Treatment
All the treatments were maintained in duplicates (n=2). Tissues were treated with the negative control, positive control followed by the test item.
8.5.1 Pre-Treatment
After the overnight incubation, the tissues were pre-wetted by adding 20 μL of Ca++Mg++Free-DPBS on to the tissue surface. The plates were gently tapped to assure that the entire tissue surface is wetted. The tissues were then incubated at 37oC, 5% Co2 for 30 minutes.
8.5.2 Test Item / Controls Exposure
After the 30 minutes Ca++Mg++Free-DPBS pre-treatment, 50 μL each of negative control, positive control and test item was applied topically on the EpiOcular™ tissues to cover the upper surface.

The tissue insert were gently tapped to make sure the test item/controls spreads all over the surface of the tissue. Tissues were incubated at 37oC, 5% Co2 for 30 minutes.
8.5.3 Rinsing
At the end of treatment time (30 minutes), controls/test item were removed by extensively rinsing the tissues with Ca++Mg++ free DPBS as described in detail below.
Three sets of sterile beaker (100 mL capacity) containing 75 mL of Ca++Mg++ free DPBS were prepared for each treatment (test item and controls tested).
The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps, two tissues of the same treatment were rinsed at a time by holding replicate inserts together by their collars using forceps. Care was taken not to damage the tissues by the forceps.
The test item and controls were decanted from the tissue surface onto a clean absorbent material and the tissues were dipped into the first beaker of DPBS, swirled in a circular motion for approximately 2 seconds, lifted out and the liquid was decanted back into the container. This process was performed for two additional times in the first beaker.
The tissues were then rinsed in the second and third beakers of DPBS three times each in the same manner. Finally, remaining liquid was decanted onto the absorbent material.
8.5.4 Post Soak
After rinsing, the tissues were immediately transferred to a pre labelled 12 well plate containing 5 mL of previously warmed assay medium, immersed and kept at room temperature for 12 minutes to facilitate the removal of any residue test item.
8.5.5 Post Incubation
At the end of the Post-Soak immersion, each tissue was removed from the assay medium, the medium was decanted off the tissue, and the tissue construct was blotted on absorbent material.
The tissues were then transferred to a pre-labeled 6 well plate containing 1 mL of warm assay medium. The tissues were incubated at 37oC, 5% Co2 for 2 hours.

8.5.6 MTT Viability Assay
Post treatment incubation of 2 hours, MTT assay was performed.
a) 300 μL of the MTT solution was added to each designated well of a pre labeled 24 well plate.
b) Each tissue 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 plates, the plates were incubated at 37oC, 5% CO2 for 3 hours.
c) After 3 hour incubation with the MTT solution, each tissue insert was removed from the 24 well plate, the bottom of the insert was blotted on absorbent material, and then transferred to a pre labeled 24 well plate containing 2 mL of isopropanol. The plates were sealed with parafilm and were stored overnight at 2-8°C in the dark. The next day, plates were kept on an orbital plate shaker for shaking at 300 rpm, 25°C for 2 hours 30 minutes to extract the MTT. At the end of the extraction period, the tissues were pierced and the liquid within each tissue insert was decanted into the well from which it was taken.
d) The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells according to the plate map. 200 µL of isopropanol was added to the wells designated as blanks. The absorbance at 570 nm of each well was read in microplate reader.

Results and discussion

In vitro

Results

Irritation parameter:

other: Percentage viability of tissue

Run / experiment:

mean of four tissue inserts (2 tissues; 2 inserts / tissue)

Value:

92.59

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

deionised water (100% viability)

Positive controls validity:

valid

Remarks:

methyl acetate (35,75% viability)

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD was in between > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean tissue viability of the positive control was < 50% compared to
the negative control.

Any other information on results incl. tables

Interpretation of results was carried out according to prediction model described below.

Prediction model

If the test item-treated tissue viability is > 60.0% relative to negative control-treated tissue viability, the test item is labeled as non-irritant.

If the test item-treated tissue viability is ≤ 60.0% relative to negative control-treated tissue viability, the test item is labeled as irritant.

In vitroresult	In vivoprediction
mean tissue viability ≤ 60.0%	Irritant
mean tissue viability > 60.0%	Non Irritant

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test method and test conditions employed the tissues treated with test item showed a relative percent viability 92.59 % (> 60 %) hence, it is concluded that the test item Cypriol (Cyperus scariosus ext. Oil) was Non-Irritant (NI).

Executive summary:

In vitro eye irritation test was carried out using Reconstructed Human Cornea-like Epithelium with an objective to evaluate Eye Irritation potential of the test item Cypriol (Cyperus scariosus ext. Oil).

Pre-checks were performed on Cypriol (Cyperus scariosus ext. Oil) to identify if the test item was a direct MTT reducers and/or colour interfering substance. Test item was found to be non- reducer of MTT and did not interfere with OD as the absorbance 570nm of test item in isopropanol was 0.0053 (not greater than 0.08).

EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories. Upon receipt, the tissues were equilibrated to room temperature for 15 minutes. Tissues were inspected for any air bubbles between the agarose gel and insert. Then the tissues were carefully removed from agarose, blotted to remove agarose sticking to the inserts and transferred into 6 well plate containing 1 mL of assay medium and incubated at 37°C, 5% Co2 for 1 hour. After the incubation period assay medium was replaced with fresh assay medium and incubated at 37°C, 5% Co2 overnight for 16 hours.

Following overnight incubation, tissues were pre-wetted with 20 μL of Ca++Mg++ Free-DPBS and incubated at 37°C, 5% Co2 for 30 minutes.

After prewetting, 50 μL each of negative control (sterile deionized water), positive control (Methyl Acetate) and test item (Cypriol) was added on to the tissue surface. All the treatments were carried in duplicates (2 EpiOcular tissues/treatment) and incubated at 37oC, 5% Co2 for 30 minutes.

Post treatment, all the tissues were made free of negative control, positive control and test item by rinsing with Ca++Mg++ free DPBS. Post rinsing, the tissues were dried by blotting on to the tissue paper and soaked in 5 mL of assay medium filled in 12 well plate and incubated further for 12 minutes at room temperature.

Post soaking in media, tissues were transferred to pre labelled 6 well plate containing 1 mL of assay medium and incubated at 37oC, 5% Co2 for 120 minutes.

After recovery period of 120 minutes, MTT assay was carried out by transferring inserts into a 24 well plate containing 0.3 mL of MTT solution and incubated at 37°C, 5% Co2 for 180 minutes. The inserts were transferred to a pre labelled 24 well plate containing 2 mL of isopropanol. The plate was sealed and stored overnight at 2-8°C in the dark and on the next day, the plates were kept on orbital shaker for 2 hours 30 minutes to extract the MTT. MTT-formazan extracted in isopropanol was quantified by optical density (OD) measurement at 570 nm. OD values were analyzed to calculate the relative percent viability of the tissues.

The relative percent viability of the tissues treated with the test item Cypriol (Cyperus scariosus ext. Oil) was 92.59 % considering the mean negative control as 100 % viability. Under the same conditions the positive control

Methyl Acetate showed only 35.75 % viability confirming the sensitivity of the test system.

Under the test method and test conditions employed the tissues treated with test item showed a relative percent viability > 60 % hence, it is concluded that the test item Cypriol (Cyperus scariosus ext. Oil) was Non-Irritant (NI).