5H-Cyclopenta[h]quinazoline, 6,6a,7,8,9,9a-hexahydro-7,7,8,9,9-pentamethyl-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 08 April 2014 and 10 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

EC Number:

801-694-5

Cas Number:

1392325-86-8

Molecular formula:

C16H24N2

IUPAC Name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

Test material form:

other: Solid

Details on test material:

Identification: IFF TM 12-206 (FRET 10-0199)
Description: White solid block
Storage conditions: Approximately 4 °C, in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- EPISKIN™ Reconstructed Human Epidermis Model Kit
- Supplier: SkinEthic Laboratories, Lyon, France
- Date received: 08 April 2014
- EpiSkinTM Tissues (0.38cm2) lot number: 14-EKIN-012
- Maintenance Medium lot number: 14-MAIN3-014
- Assay Medium lot number: 14-ESSC-013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.

TEST FOR DIRECT MTT REDUCTION
20 mg of the test item was added to 2.0 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turned blue, the test item is presumed to have reduced the MTT and the determination of skin corrosion potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.
The test item was shown to directly reduce 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 water killed tissues.
This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues.
Water-killed tissues were prepared by placing untreated EPISKINTM tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, described in the main test, the MTT reducing test item was applied to one water killed tissue for each exposure period. In addition, one water killed tissue for the 240-Minute exposure time remained untreated. The untreated water killed tissue demonstrates a small amount of MTT reduction due to residual reducing enzymes within the tissue.

PRE-INCUBATION (DAY 0: TISSUE ARRIVAL)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:

Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes

2.0 mL of maintenance medium, warmed to approximately 37 ºC, was pipetted into two wells of the first column of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12 well plate was used for each test item, control and time point. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

MAIN TEST - APPLICATION OF TEST ITEM AND RINSING (DAY 1)
2.0 mL of assay medium, warmed to approximately 37 °C , was pipetted into 2 wells of the second and third columns of the 12 well plate. The tissues were transferred into the second column.

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 20 mg of the test item was applied topically to the corresponding tissues and 100 µl of 0.9% w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 µl of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 µl of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
2.0 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12-well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for approximately 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labeled 1.5 mL micro tubes containing 500 µl of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 2)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using an Anthos 2001 microplate reader.


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if after 3 minutes the relative mean tissue viability is <35%, or if the viability after 3 minutes exposure is greater than or equal to 35 % and the viability after 1 hour exposure is less than 35%, or if the viability after 1 hour exposure is greater than or equal to 35% and the viability after 4 hour exposure is less than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hour exposure is greater than 35%.

QUALITY CRITERIA:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative control: The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is > 0.600 and <1.500.
Positive control: The assay established the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20% relative to the negative control treated tissues following the 240-minute exposure period.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

VEHICLE : 0.9% w/v sodium chloride for wetting the test item
- Amount(s) applied (volume or weight with unit): 100 µL

NEGATIVE CONTROL : 0.9% w/v sodium chloride
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL : glacial acetic acid
- Amount(s) applied (volume or weight): 50 µL

Duration of treatment / exposure:

3, 60 and 240 minutes

Number of replicates:

2 for the test item, the negative and the positive control, each.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minutes exposure

Value:

128.9

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

127.2

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

111.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Results (see table 1, 'Any other inormation on results, incl. tables)
- The relative mean viabilities of the test item treated tissues were:
240 minutes exposure: 128.9%
60 minutes exposure: 127.2%
3 minutes exposure: 111.5%


Test for direct MTT reduction:
- The MTT solution containing the test item turned blue. This was taken to indicate that the test item reduced MTT and the MTT viability assay was therefore performed in parallel on viable and water-killed tissues. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

Quality criteria:
- The relative mean tissue viability for the positive control treated tissues was 6.0% relative to the negative control treated tissues following the 240-minute exposure period.
- The mean OD562 for the negative control treated tissues was 0.871.
- As both quality criteria were met, the test is considered valid.

Any other information on results incl. tables

Table 1 Mean OD₅₆₂ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Period	Mean OD562of individual tissues	Mean OD562of duplicate tissues	Relative mean viability (%)
Negative Control Item	240 Minutes	0.952	0.871	100*
		0.790
Positive Control Item	240 Minutes	0.064	0.052	6.0
		0.039
Test Item	240 Minutes	1.054	1.123	128.9
		1.191
	60 Minutes	1.118	1.108	127.2
		1.098
	3 Minutes	1.020	0.971	111.5

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

Applicant's summary and conclusion

Interpretation of results:

other: Non-Corrosive

Remarks:

in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance does not cause skin corrosion.

Executive summary:

The substance was tested in vitro for skin corrosion in an EPISKIN Reconstructed Epidermis Model (OECD TG 431). Skin tissue was tested for direct MTT reduction before the start of the test. The substance was tested in duplicate on skin tissue at an amount of 20 mg. Sodium chloride (0.9% (w/v)) was used as vehicle. Sodium chloride (0.9% (w/v)) and glacial acetic acid were tested in duplicate as negative and positive control, respectively. Tissues were treated with the substance for an exposure time of 3, 60 and 240 minutes. The substance showed direct MTT reduction and therefore the viability assay was performed in parallel on viable and water-killed tissues. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT and correction of the results was considered unnecessary. The relative mean viabilities of the test substance treated tissues were: 111.5%, 127.2% and 128.9% for the 3, 60 and 240 minutes exposure, respectively. Based on these findings, the substance is not corrosive to the skin.