2,5-di-tert-butyl-p-benzoquinone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Sep 2017- 28 Nov 2017

Reliability:

1 (reliable without restriction)

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,5-di-tert-butyl-p-benzoquinone

EC Number:

219-552-0

EC Name:

2,5-di-tert-butyl-p-benzoquinone

Cas Number:

2460-77-7

Molecular formula:

C14H20O2

IUPAC Name:

2,5-di-tert-butyl-1,4-benzoquinone

Test material form:

solid: bulk

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The test system used iwas Reconstructed Human Cornea-like Epithelium (RhCE). RhCE cell construc, EpiOcular TMt is a nonkeratinized epithelium composed of normal human keratinocytes in a three-dimensional structure. It mimics the corneal epithelium with progressively stratified but not cornified cells.EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Slovakia. Cat no. OCL-200-EIT. Lot no. 27005.

Test system

Vehicle:

other: MTT

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg test item in 1 ml of MTT

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

18 hours

Details on study design:

Test Material Exposure (Day 1)
a. Test item exposure: After the 30±2 minutes Ca++Mg++Free-DPBS pretreatment, 50 μL each of negative control, positive control and 50 mg of test item tested by applying topically on the EpiOcular™ tissues so as to cover the upper surface.
b. 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 37±1oC, 5% CO2 and humidified atmosphere for 6 ± 0.25 hours.

Rinsing
At the end of treatment time (6 ± 0.25 hours), controls/test item were removed by extensively rinsing the tissues with Ca++Mg++-free D-PBS (brought to room temperature), as described in detail below.
Three sets of sterile beaker (150 mL capacity) containing 100 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 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, any remaining liquid was decanted onto the absorbent material.


MTT Viability Assay
Post treatment incubation of 18 ± 0.25 hours, MTT assay was performed.
a) 300 μL of the MTT solution (1 mg/mL) 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 for 180±10 minutes at Standard Culture Conditions.
c) Each tissue insert was removed from the 24-well plate after 180 ± 10 minutes; the bottom of the insert was blotted on absorbent material, 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. Plates were placed on an orbital plate shaker and shaken for 2 hours at room temperature. At the end of the extraction period, the tissues were not 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.

After rinsing, the tissues were immediately transferred and immersed in 5 mL of previously warmed assay medium (room temperature) in a pre-labeled 12-well plate for 25 ± 2 minutes at room temperature to facilitate the removal of any residue test item.

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 the appropriate wells of pre-labeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 ± 0.25 hours at standard culture conditions.

Calculation for Viability
Percentage viability was calculated for two relating tissues of controls and test item relative to the negative control (100 %). Viability % = corrected test item OD corrected mean negative control OD x 100
If the difference between two relating tissues is >20% the test is considered as non-qualified. Mean test item viability was calculated and the test item was classified according to the prediction model ( Refer section 8.5).

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

The mean tissue viability (%)

Value:

124

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

2.15

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Acceptance Criteria
• The negative control OD was in between > 0.8 and < 2.5.
• The mean tissue viability of the positive control was < 50% compared to the negative control.
• The variability between tissue replicates of test item and control substances were within the accepted limits.
o The difference of viability between two tissue replicates was less than 20% for negative control, test item and positive control treated tissue

Any other information on results incl. tables

Interpretation of results and prediction model

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 controltreated tissue viability, the test item is labeled as non-irritant.

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

Table 1. Tissue viabilities.

Item	Tissue Viability 1			Viability Tissue 2			Mean 1 & 2	Diff 1 &2	Classification
	Ali 1	Ali 2	Mean 1	Ali 1	Ali 2	Mean 2
Negative control	97,82	96,18	97,00	103,07	102,93	103,00	100,00	6,00	Non-irritant
Positive control	40,14	39,28	39,71	36,54	38,58	37,56	38,63	2,15	Irritant
Test item	119,11	118,02	118,57	128,27	130,84	129,56	124,06	10,99	Non-irritant

Ali: Aliquot, Diff: difference of viability between 2 tissues.

Table 2. OD values of individual epiocular tissues.

Item	OD570 nm Values
	Tissue 1		Tissue 2
	R1	R2	R1	R2
Negative control	1,0586	1,0416	1,1133	1,1118
Positive control	0,4586	0,4494	0,4208	0,4421
Test item	1,2802	1,2689	1,3756	1,4023

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Test item-treated tissues showed viability of > 60.0% relative to negative control-treated tissue viability, thus the test item 2, 5- di- tert-butyl -,4- benzoquinone (YAPOX 2255) was concluded to be a non-irritant (NI).

Executive summary:

A guideline compliant eye irritation study (OECD 492) –in vitro eye irritation in Reconstructed Human Cornea-like Epithelium (RhCE)) was conducted. Aim of the study was to examine the acute eye irritation potential of the test item by measurement of its cytotoxic effect on the EpiOcular™ cornea epithelial model. The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek).

 

In this assay, the test item was applied to the surface of the cornea epithelial construct for 30 minutes. After exposure period, each tissue was extensively rinsed, and the tissue was allowed to express the resulting damage. Two construct tissues were used for the treatment with test item and for negative and positive controls. The MTT assay was performed to determine tissue viability. The tissues were transferred in the 24-wells plate containing MTT medium and incubated in a humidified atmosphere for 3 hours. After incubation the blue formazan salt formed was extracted in isopropanol and the optical density of extracted formazan was determined using a spectrophotometer at 540 nm. Validity of the test method was ascertained by positive control (methyl acetate) and negative control (deionized sterile water). The tissue viability met the acceptance criterion as the mean OD of negative control was in between > 0.8 and < 2.5. The positive control met the acceptance criterion as the viability of culture treated by methyl acetate was 39% (less than 60%).The viability of culture treated by 2,5 -di-tert-butyl-1,4 -benzoquinone was 124 %. Therefore, 2,5 -di-tert-butyl-1,4 -benzoquinone

is considered to be non-irritant to the eye.

Classification of test items as irritants and non-irritants was carried according to prediction model described in test guideline OECD 492. As the test item-treated tissues showed viability of > 60.0% relative to negative control-treated tissue viability, the test item, 2,5- di-tert-butyl-1,4 -benzoquinone, was concluded to be a non-irritant (NI).