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

Endpoint summary

Description of key information

2,5 -di-tert-butyl-p-benzoquinone was tested for its possible skin corrosion potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application. The test was performed using OECD 431 guideline. After 3 min, 60 min and 240 min exposures with the test item, the observed cell viability was approximately 100%.

The study indicate that the test item 2,5 -di tertiary butyl 1,4 -benzoquinone is non-corrosive in this in vitro skin corrosion test using reconstructed human epidermis under the conditions of testing employed.

The substance was tested for its possible skin irritation potential using a three-dimensional Reconstructed Human Epidermal model, EpiSkin. The mean relative tissue viability for 2,5-di-tert-butyl-1,4-benzoquinone was above 50% and hence it is non-irritant under the experimental conditions described.

Eye irritation was potential was investigated 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).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Aug 2017- 21 Nov 21 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

EpiSkin kit from SkinEthic Laboratories, France.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

Source of the test system: SkinEthic Laboratories, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 mg

Duration of treatment / exposure:

3, 60 and 240 min

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

153.15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

105.75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minute exposure

Value:

94.701

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1. OD values of individual epidermis units, 3 min exposure

	Absorption, OD570
	R1	R2
Negative control	0,49600	0,49125
Test item	0,75120	0,76080
Negative control (color)	0,00460	-
Non-specific color control	0,00480

able 2. OD values of individual epidermis units, 60 min exposure

	Absorption, OD570
	R1	R2
Negative control	0,61720	0,61470
Test item	0,65080	0,65190
Negative control (color)	0,00585	0,00550
Non-specific color control	0,00565	0,00640

able 3. OD values of individual epidermis units, 240 min exposure

	Absorption, OD570
	R1	R2
Negative control	0,54845	0,5480
Positive control	0,02695	0,02180
Test item	0,52150	0,51685
Negative control (color)	0,00225	0,00190
Non-specific color control	0,00205	0,00350

Interpretation of results:

GHS criteria not met

Conclusions:

The test item 2,5-di tertiary butyl 1,4-benzoquinone is non-corrosive in the EpiSkin in vitro skin corrosion model.

Executive summary:

The test item was tested for its possible skin corrosion potential using a three-dimesional Reconstructed Human Epidermis model, EpiSkin, through topical application. After 3 min, 60 min and 240 min exposures with the test item, the observed cell viability was 153% and 106% and 95%, respectively.

The positive control had a mean cell viability of 4.44% after 240 -minutes exposure. The absolute mean OD570 of the negative control tissue was 0.493625, 0.61595 and 0.548225 for the 3 -minute, 60 -minutes and 240 -minutes exposures, respectively. The results of the controls indicate that the test system functioned properly.

The study indicate that the test item 2,5 -di tertiary butyl 1,4 -benzoquinone is non-corrosive in this in vitro skin corrosion test using reconstructed human epidermis under the conditions of testing employed.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Aug 2017-24 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin from SkinEthic Laboratories, Lyon, France. Batch nro. 17-EKIN-044. After receipt, 13 epidermis units were transferred to 12-well plates containing 2 ml of pre-warmed maintenance medium and incubated in a CO2 incubator for 1 hour 15 minutes.
Cell viability was assessed by measuring mitochondrial activity. The tissues were incubated with MTT solution. MTT was reduced by succinated dehydrogenase into a blue formazan precipitate in the mitochondria of living cells. Prior to treatment in the irritation assay, the test item was evaluated for its intrinsic color or ability to become colored in contact with water. Prior to treatment in the irritation assay, the test item was checked for possible direct MTT reduction by exposing it in direct contact with the MTT solution.
After exposures the epidermal units were dried carefully and prefilled with MTT-solution and plated were incubated for 3 hours at 37oC in a carbon dioxide incubator with 5% CO2. After incubation with MTT, the epidermis units were placed on an absorbent paper to dry the tissues. Total biopsy was made by using a biopsy puch. Epidermis was separated from the collagen matrix using forceps and bothe parts were placed in pre-labaled microtubes and extracted with 500 ul of acidic isopropanol. Tubes were stored at room temperature for 4 hours.
About 200 ul sample from each tube was transferred into the wells of a labeled 96-well flat bottom plated (2 wells/epidermis unit) and the amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicated with TECAN Infinite M200 Microplated reader, using acidified isopropanol solution as the blank.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value

Value:

133

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The absolute mean OD570 of the negative control tissues was 0,5694. The positive control has a mean cell viability of 0,03865% after 15 minutes exposure, indicating that the test system functioned properply.

Table 1. Mean OD values of individual epidermis units.

	Absorption (OD570)
	R1	R2	R3
Negative control	0,6064	0,6114	0,61355
Positive control	0,0771	0,0799	0,0821
Test item	0,78185	0,80515	0,80305
Negative control (color)	0,0449	0,0447	-
Non-specific color control	0,0494	0,0586	-

Blank (isopropanol) OD value (mean of 6 replicate values): 0.04105.

OD: optical density

Table 2. True OD values of individual epidermis units.

	Absorption (OD570)
	R1	R2	R3	Mean	SD
Negative control	0,56535	0,57035	0,5725	0,5694	0,0036
Positive control	0,03605	0,03885	0,04105	0,038565	0,003
Test item	0,7408	0,7641	0,762	0,75563	0,013
Negative control	0,00385	0,00365	-	0,00375	-
Nonspecific color control	0,00835	0,01735	-	0,01295	0,007

OD= optical density

True OD value= OD Raw-OD Blank

Note: the OD values of the concurrent negative control tissues set up along with NSC control were not used in calculation.

Table 3. Individual tissue viability of epidermis units.

	% Individual Viability
	R1	R2	R3	Mean	SD
Positive control	6,33	6,82	7,21	6,79	0,441
Test item	130,10	134,19	133,82	132,70	2,262
Non-specific color control	1,46	3,08	-	2,27	1,146

Negative control mean: 0,5694

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative tissue viability for the test item, 2,5-di-tert-butyl-1,4-benzoquinone was above 50% and hence it is non-irritant under the experimentl conditions described.

Executive summary:

The test item, 2,5 -di-tert-butyl-1,4 -benzoquinone was tested for its possible skin irritation potential using a three-dimensional Reconstructed Human Epidermal model, EpiSkin, through topical application for 15 minutes.

The test item is in solid form and was applied directly on the top of the skin tissues at 10 mg/tissue, along with 5ul of distilled water and exposed for 15 minutes.

Ten microliters (10 ul) pf PBS and 10 ul of 5% aqueous SDS used as the negative and positive controls, respectively.

After approximately 42 -hour post-incubation perid, irritation potential of 2,5 di-tert-butyl-1,4 -benzoquinone was evaluated by assessing the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 minutes treatment with the test item was compared to the negative control tissues. The mean relative tissue viability for the test item, 2,5-di-tert-butyl-1,4-benzoquinone was above 50% (133%) and hence it is non-irritant under the experimentl conditions described.

The absolute mean OD570 of the negative control tissues was 0.5694. The positive control has a mean cell viability of 0.0385% after 15 minutes exposure, indicating that the test system functioned properly.

The study indicated that the test item 2,5-di-tert-butyl-1,4-benzoquinone is non-irritant in in vitro skin irritation test using Reconstructed Human Epidermis under the conditions of testing employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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)

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)

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.

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).

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

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

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The results of the key study studies do not indicate the substance to be classified for skin irritant according to CLP Regulation 1272/2008.

The result of the key study does not indicate the substance to be classified for eye irritant according to CLP Regulation 1272/2008.