3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

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 23 March 2016 and 25 March 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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

EC Number:

814-345-7

Cas Number:

2003244-43-5

Molecular formula:

C14H21N

IUPAC Name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Details on test material:

- Name of test material (as cited in study report): ES421 Pinyl Nitrile- Molecular weight (if other than submission substance): 203 g/mol- Physical state: Crystallized white powder- Analytical purity: 98.08% (per Certificate of Analysis)- Composition of test material, percentage of components: 2-Norpinene-2-propionitrile, alpha, alpha,6,6-tetramethyl- Purity test date: 08 February 2016- Lot/batch No.: SM15077102- Storage condition of test material: 2-8°C, protected from light

Specific details on test material used for the study:

dentification: ES421 Pinyl Nitrile

SOURCE OF TEST MATERIAL
- Appearance/physical state: white solid
- Source and lot/batch No.of test material: SM15077102
- Expiration date of the lot/batch: 22 July 2016
- Purity: 98.08%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °C, in the dark

The test item was warmed to 45 °C in a water bath prior to dosing.

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™ Reconstructed Human Epidermis Model Kit

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Supplier MatTek

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System: EpiDerm™ Reconstructed Human Epidermis Model Kit

Supplier: MatTek
Date received: 22 March 2016
EpiDermTM Tissues (0.63cm2) lot number: 23325
Assay Medium lot number: 031716TMA
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 relative to the control, the test item was presumed to have reduced the MTT. 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 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 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.

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 were placed into a refrigerator overnight, 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

The test item was shown to directly reduce MTT and freeze-killed tissues were employed, the results of the MTT assay were therefore corrected as follows:
True viability = mean OD tvt−(OD tkt−OD ukt)
OD = optical density at 562 nm
tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues

If direct reduction by the test item is greater than 30% of the negative control value, additional steps must be taken into account or the test item may be considered incompatible with this test system. If direct reduction by the test item is less than 30% of the negative control value, the mean OD of the test item treated killed control may be 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.

Classification of corrosivity potential is based on relative viabilities for both exposure times. Refer to '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 8.0 N 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

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

NEGATIVE CONTROL
Identification: Sterile distilled water
Batch: 3011529
Purity: Not supplied
Expiry Date: 01 February 2017
Storage Conditions: Room temperature

POSITIVE CONTROL
Information as provided by the Supplier.
Identification: 8.0 N Potassium Hydroxide
Batch: SLBJ7763V
Purity: 8.0N
Expiry Date: 05 June 2016
Storage Conditions: Room temperature

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 117.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item was considered to be non-corrosive to the skin.

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

ca. 120.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item was considered to be non-corrosive to the skin.

Any other information on results incl. tables

Test Item, Positive Control Item and Negative Control Item

 

Mean OD₅₆₂values and viabilities for the negative control, positive control and test item are given below:

Tissue	Exposure Period	MeanOD562of individual tissues	Mean OD562of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.609	1.467	0.202	13.7	100*
		1.324
	60 Minutes	1.945	1.781	0.233	13.1
		1.616
Positive Control	3 Minutes	0.057	0.061	0.005	na	4.1
		0.064
	60 Minutes	0.064	0.067	0.004	na	3.8
		0.070
Test Item	3 Minutes	1.753	1.728	0.035	2.0	117.8
		1.703
	60 Minutes	1.970	2.153	0.258	12.0	120.9
		2.335

O=   Optical density

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

na=  Not applicable

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	4.1	117.8
60 minute	100*	3.8	120.9

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

Quality Criteria

 

The mean OD₅₆₂for the negative control treated tissues was 1.467 for the 3-Minute exposure period and 1.781 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 3.8% 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.

Applicant's summary and conclusion

Interpretation of results:

other: The test item was considered to be non-corrosive to the skin.

Conclusions:

The test item, ES421 Pinyl Nitrile, was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item, ES421 Pinyl Nitrile, using the EpiDerm™ Human Skin Model (after OECD Guideline for the Testing of Chemicals No. 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method, 28 July 2015) after treatment periods of 3 and 60 minutes with ES421 Pinyl Nitrile. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

 

Methods

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 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 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-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD₅₆₂). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	4.1	117.8
60 minute	100*	3.8	120.9

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

Quality criteria

The quality criteria required for acceptance of results in the test were satisfied.