Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-08-2018 to 21-08-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2017 ; signature: November 2017

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran

EC Number:

915-782-7

Molecular formula:

C12H14O

IUPAC Name:

Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, under Nitrogen, in the dark
- Other: Colourless (sponsor) ; clear colourless (laboratory)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): Lot no.: 28646
- Production date: Not reported.
- Shipping date: Not reported.
- Delivery date: 21-08-2018
- Date of initiation of testing: ca. 22-08-2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, 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.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT (prepared from a MatTek MTT-100 kit)
- Incubation time: The plate was incubated (37 °C, 5% CO2) for 3 hours.
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm (OD570)
- Filter: Not reported.
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 4.6% 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.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Two (2), duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: The MTT solution containing the test item turned blue. Therefore, an assessment found the test item was able to directly reduce MTT and an additional procedure using freeze-killed tissues was performed. The results of the freeze-killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
- Procedure used to prepare the killed tissues (if applicable): Freeze-killed ; Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM 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.
- N. of replicates : Two (2), duplicate
- Method of calculation used: Interference by the test item relative to the corresponding negative control:
(a) 3 minutes exposure:
Mean of test item treated killed tissues (tkt) = 0.170 OD570
Mean of untreated killed tissues (ukt) = 0.227 OD570
The direct reduction by the test item relative to the negative control value:
(0.170 (tkt) – 0.227 (ukt)) / 1.796 (mean of negative control) = 0.0%*
*Negative value treated as 0.0%
(b) 60 minutes exposure:
Mean of test item treated killed tissues (tkt) = 0.257 OD570
Mean of untreated killed tissues (ukt) = 0.193 OD570
The direct reduction by the test item relative to the negative control value:
(0.257 (tkt) – 0.193 (ukt)) / 1.885 (mean of negative control) = 3.4%
The interference by the test item relative to the corresponding negative control was 0.0% after 3 minutes exposure and 3.4% after 60 minutes exposure. Therefore direct reduction was < 30% relative to the negative control and considered acceptable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Cut off points in accordance with OECD TG 431 and the GHS and CLP Classification systems.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test substance at exposure times 3-minutes and 60-minutes, respectively. Where necessary, direct MTT reduction, colour interference and correction for background Isopropanol absorbance at OD570 (via measurement of triplicate blanks) was completed.

OTHER:
EpiDerm Skin Model (EPI-200, Lot no.: 28646). The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EpiDerm Skin Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Preincubation:
Upon receipt of the Epiderm tissues, the sealed 24-well plate was stored in a refrigerator until use. The assay medium was brought to room temperature 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:
- 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.
- The liquid test item was applied directly on top of the skin tissue. 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 sterile distilled water (negative control) was added to the first two tissues. 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
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, 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.
- After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labelled 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of the test item
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl sterile distilled water (negative control)
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8N KOH(aq) (pre-diluted)

Duration of treatment / exposure:

Observations are made 3-minutes and 1-hour post-test item application

Duration of post-treatment incubation (if applicable):

After rinsing: The plate was incubated (37 °C, 5% CO2) for 3 hour MTT incubation. Subsequently, the inserts were blotted and transferred to labelled 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.

Number of replicates:

Two (n=2) for a 3-minute exposure to the test item and two for a 60-minute exposure

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 minute exposure

Run / experiment:

mean (n=2)

Value:

112.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 3 minute exposure. Remarks: n=2 ; CV = 0.2% ; Score in terms of percentage of negative control.

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 minute exposure

Run / experiment:

mean (n=2)

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 60 minute exposure. Remarks: n=2 ; CV = 7.2% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Corrected for by use of freeze-killed tissue replicates. The interference by the test item relative to the corresponding negative control was 0.0% after 3 minutes exposure and 3.4% after 60 minutes exposure. Therefore direct reduction was < 30% relative to the negative control and considered acceptable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Positive Control and Negative Control HCD data are presented within the full study report and the concurrent NC and PC was within the specified ranges.

Any other information on results incl. tables

Table 1. Mean absorption and tissue viability in the in vitro skin corrosion test with the test item

Tissue	Exposure Period	MeanOD570 of individual tissues (tvt)	Corrected Mean OD570 (tvt-[tkt-ukt])	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.755	-	1.796	0.057	3.2	100*
		1.836	-
	60 Minutes	1.910	-	1.885	0.036	1.9
		1.859	-
Positive Control	3 Minutes	0.092	-	0.081	0.016	na	4.5
		0.069	-
	60 Minutes	0.097	-	0.087	0.014	na	4.6
		0.077	-
Test Item	3 Minutes	2.013	2.013	2.017	0.005	0.2	112.3
		2.020	2.020
	60 Minutes	1.818	1.754	1.848	0.132	7.2	98.0
		2.005	1.941

OD = Optical density

tvt = Treated killed tissues

tkt = Untreated killed tissues

ukt =Untreated killed tissues

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

na = Not applicable

 

3 minute exposure corrected mean OD570:

x̅ (0.163 + 0.176) = 0.170 (tkt) – x̅ (0.226 + 0.228) = 0.227 (ukt) = 0.000+
+Negative value treated as 0.0% to avoid artificially inflating the results.

60 minute exposure corrected mean OD570:

x̅ (0.280 + 0.233) = 0.257 (tkt) – x̅ (0.178 + 0.207) = 0.193 (ukt) = 0.064

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test item is not considered to be corrosive in the in vitro skin corrosion test using EpiDerm Skin Model.

Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS under GLP to assess the skin corrosion potential of the test item using a human three dimensional epidermal model EpiDerm (EPI-200). Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes together with a negative control and positive control. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570). The mean OD570 for the negative control treated tissues was 1.796 for the 3 Minute exposure period and 1.885 for the 60 Minute exposure period. The relative mean tissue viability obtained after the 3-minute and 60-minute treatments with the test item (corrected for direct MTT reduction) compared to the negative control tissues was 112.3% and 98.0% respectively. The relative mean tissue viability for the positive control after the 3-minute and 60-minute treated tissues compared to the negative control tissues was 4.5% and 4.6%, respectively. The quality criteria required for acceptance of results in the test were satisfied. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 60-minute treatment the test item is not considered to be corrosive. Under the conditions of this study, the test item is not corrosive in the in vitro skin corrosion test.