Benzeneacetaldehyde, cyclic acetal with glycerol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2016 to 13 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Benzeneacetaldehyde, cyclic acetal with glycerol

EC Number:

249-934-2

EC Name:

Benzeneacetaldehyde, cyclic acetal with glycerol

Cas Number:

29895-73-6

Molecular formula:

C11H14O3

IUPAC Name:

Reaction mass of cis-2-benzyl-1,3-dioxan-5-ol and rel-[(2R,4R)-2-benzyl-1,3-dioxolan-4-yl]methanol and rel-[(2R,4S)-2-benzyl-1,3-dioxolan-4-yl]methanol and trans-2-benzyl-1,3-dioxan-5-ol

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.

Cell source:

other: MatTek

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
EpiDerm™ Reconstructed Human Epidermis Model Kit:
Supplier: MatTek
Date received: 10 May 2016
EpiDermTM Tissues (0.63cm2) lot number: 23335
Assay Medium lot number: 050516TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

STUDY DESIGN
PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT:
MTT Dye Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turns blue or purple relative to the control, the test item was presumed to have reduced the MTT.
The test item was found to be a possible direct reducer of MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze- killed tissues.
This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
Freeze-killed tissues were prepared by placing untreated EPIDERM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the MTT reducing test item was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated. The untreated freeze killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

MAIN TEST
Pre-Incubation:
The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing:
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 µL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure. For the 60-Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.
When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements:
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

DATA EVALUATION
Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100

Classification of corrosivity potential is based on relative viabilities for both exposure times according to the table presented under Any other information on materials and methods incl. tables.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control: The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control: Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
Coefficient of Variation: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3-Minute exposure period and 60-Minute exposure period

Duration of post-treatment incubation (if applicable):

3 hours MTT incubation period and overnight MTT extraction period

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-Minute exposure period

Value:

87.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Relative mean tissue viability compared to the negative control tissues (viability set at 100%)

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-Minute exposure period

Value:

67.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Relative mean tissue viability compared to the negative control tissues (viability set at 100%)

Other effects / acceptance of results:

Direct MTT Reduction:
The test item was found to be a possible direct reducer of MTT. Therefore, an additional procedure using freeze-killed tissues was performed during the determination of skin corrosion potential as a precaution. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results. The results generated from the freeze-killed tissues are presented below.

3 minutes exposure:
Mean of test item treated killed tissues (tkt) = 0.147 OD562
Mean of untreated killed tissues (ukt) = 0.139 OD562
The direct reduction by the test item relative to the negative control value:
(0.147 (tkt) – 0.139 (ukt)) / 1.858 (mean of negative control) = 0.4%

60 minutes exposure:
Mean of test item treated killed tissues (tkt) = 0.143 OD562
Mean of untreated killed tissues (ukt) = 0.136 OD562
The direct reduction by the test item relative to the negative control value:
(0.143 (tkt) – 0.136 (ukt)) / 1.669 (mean of negative control) = 0.4%

Assessment of Color Interference with the MTT endpoint:
The solution (sterile water) containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test Item, Positive Control Item and Negative Control Item:
Mean OD562 values and viabilities for the negative control, positive control and test item are given under Any other information on results incl. tables.

Quality Criteria
The mean OD562 for the negative control treated tissues was 1.858 for the 3-Minute exposure period and 1.669 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 4.5% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD562of individual tissues	Mean OD562of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.030	1.858	0.243	13.1	100*
		1.686
	60 Minutes	1.676	1.669	0.010	0.6
		1.662
Positive Control	3 Minutes	0.096	0.094	0.003	na	5.1
		0.092
	60 Minutes	0.086	0.075	0.016	na	4.5
		0.063
Test Item	3 Minutes	1.705	1.630	0.106	6.5	87.7
		1.555
	60 Minutes	0.994	1.128	0.189	16.7	67.6
		1.261

OD =Optical density

*=    The mean % viability of the negative control tissue is set at 100%

na=  Not applicable

Applicant's summary and conclusion

Interpretation of results:

other: Non-corrosive to the skin

Remarks:

in accordance with EU CLP and its amendents (1272/2008 and its amendments)

Conclusions:

The test item was considered to be non-corrosive to the skin, because the viability exceeded 50% after 3 min exposure (87.7%) and 15% after 60 min exposure (67.6%).

Executive summary:

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model according to OECD TG 431 and GLP principles. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to be a possible direct reducer of MTT and therefore additional freeze-killed tissues were incorporated into the testing for correction purposes. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred (direct reduction by the test item relative to the negative control value is 0.4%). It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results. The relative mean viability for test item treated tissues after 3-Minute and 60-Minute exposure period was 87.7% and 67.6%, respectively. The test item was considered to be non-corrosive to the skin, because the viability exceeded 50% after 3 min exposure and 15% after 60 min exposure. The mean OD562 for the negative control treated tissues after 3-Minute and 60-Minute exposure period was 1.858 and 1.669, respectively (acceptance criteria ≥ 0.8 and ≤ 2.8). The relative mean tissue viability for the positive control treated tissues was 4.5% relative to the negative control following the 60-Minute exposure period (<15%). In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group ranged between 0.6 and 16.7% (=<30%). The quality criteria required for acceptance of results in the test were satisfied.