5-octylbicyclo[2.2.1]hept-2-ene

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2018 to 15 February 2018 (Laboratory Phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-octylbicyclo[2.2.1]hept-2-ene

EC Number:

814-965-8

Cas Number:

22094-84-4

Molecular formula:

C15H18 C15H26

IUPAC Name:

5-octylbicyclo[2.2.1]hept-2-ene

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Materia., Inc.
- Lot No.of test material: RP367_ONB-D
- Expiration date of the lot/batch: 9 August 2018
- Purity test date: 9 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from exposure to light
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: Test item administered as supplied. No stabilitlity of the test item in a solvent performed.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item administered as supplied

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™ Skin Model (EPI-200)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human epidermis

Justification for test system used:

The purpose of this study was to assess the potential skin corrosivity of the test article, 5-Octyl-2-norbornene, supplied by Materia, Inc., in the EpiDerm Kit (MatTek Corporation). The protocol was consistent with OECD 431 “In Vitro Skin Corrosion: Human Skin Model Test” .

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm EpiDerm™ Skin Model
- Tissue batch number: Not specified
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 13 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: For 3-minute test item exposure tissues were held at room temperature, while cultures exposed for 60 minutes were incubated in the dark at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions).
- Temperature of post-treatment incubation (if applicable): Not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
Three hundred (300) µL of 1 mg/mL MTT reagent solution was added to designated wells in a pre-labeled 24-well plate. Plates were held in an incubator until tissues were added. After the appropriate exposure time, the EpiDerm™ tissues were rinsed with warm (approximately 37ºC) Calcium and Magnesium-Free Dulbecco's Phosphate Buffered Saline (Ca++Mg++-Free DPBS) and the wash medium decanted. The EpiDerm™ tissues were transferred to the appropriate wells after rinsing. The plates were incubated at standard culture conditions for 3 ± 0.1 hours.

After the incubation period with MTT solution, the EpiDerm™ tissues were blotted on absorbent paper, cleared of excess liquid, and transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were covered with paraffin film and stored in the refrigerator (2-8ºC) until the last exposure time was harvested. Then the plates were shaken for 2 - 3 hours at room temperature.

At the end of the extraction period, the liquid within the cell culture inserts was decanted into the well from which the cell culture insert was taken. The extract solution was mixed and 200 µL was transferred to the appropriate wells of a 96-well plate. Two hundred (200) µL of isopropanol was placed in the two wells designated as the blanks. The absorbance at 550 nm (OD550) of each well was measured with a Molecular Devices Vmax plate reader.

- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 ± 0.1 hours.
- Spectrophotometer: Molecular Devices Vmax plate reader
- Wavelength: 550 nm
- Filter: Not specified
- Filter bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: yes, the assay was accepted as the positive control resulted in a corrosive classification (i.e., <50% cell viability compared to negative controls, after a 3-minute exposure and/or <15% cell viability compared to negative controls after a 60-minute exposure); and if the mean OD550 value of the negative control tissues was ≥ 0.8 and < 2.8.
- Barrier function: Yes
- Morphology: As per EpiDermTM Kit (MatTek Corporation), in accordence with OECD 431
- Contamination: None
- Reproducibility: yes, within aceptable parameters

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues : The positive control, 8N potassium hydroxide (8N KOH), is known to directly reduce MTT in the absence of viable cells. Therefore, a killed control experiment was performed concurrently in the definitive assay to determine the extent of the direct MTT reduction (if any) by the positive control in non-viable freeze-killed tissues.
- Procedure used to prepare the killed tissues (if applicable): To evaluate whether residual test article was binding to the tissue and leading to a false MTT reduction signal, a functional check (using freeze-killed control tissue) was performed. Freeze-killed tissues were received from MatTek Corporation, and were stored in the freezer until use. For the positive control, 8N KOH, duplicate killed tissues were treated in the normal fashion for 3 and 60 minutes. The rinsing, MTT exposure, and solvent extraction procedures were performed exactly as described for the viable tissues. Duplicate killed-control tissues were treated with the negative control for 3 and 60 minutes. A small amount of MTT reduction is expected from the residual NADH and associated enzymes within the killed tissue. This background reduction of MTT will be compared to the MTT reduction observed in the test article-treated killed-control tissues using calculations described in the Presentation of Data.

- No. of replicates: 2
- Method of calculation used: Since killed controls (KC) were used, additional calculations were performed to correct for the amount of MTT reduced directly by positive control residues. The raw OD550 value for the negative control killed control was subtracted from the raw OD550 values for the positive control-treated killed controls (at both exposure times), to determine the net OD550 values.

Net OD550 positive control = raw OD550 positive control KC – raw OD550 negative control KC

The net OD550 values represent the amount of reduced MTT due to direct reduction by positive control residues. The net OD550 values were subtracted from the corrected mean OD550 values of the viable positive control-treated EpiDerm™ tissues, to obtain a final corrected OD550 value.

Final corrected OD550 = corrected positive control OD550 (viable) – net OD550 positive control (KC)

Iindividual viability values were calculated as follows:
% Viability = Final corrected OD550 of Test Substance or Positive Control Exposure time / Average corrected mean OD550 of 3 & 60-minute of Negative Controls (x100)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is <50%, or if the viability after 3 minutes exposure is ≥50 % and the viability after 1 hour exposure <15%.]
- The test substance is considered to be non-corrosive to skin if viability after 3 minutes exposure is ≥50% and the viability after 1 hour exposure is ≥15%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 50µL test article (as supplied)

NEGATIVE CONTROL
- Amount(s) applied (volume): 50µL Sterile, deionized water

POSITIVE CONTROL
- Amount(s) applied (volume):50µL 8N potassium hydroxide
- Concentration (if solution): 8N KOH

Duration of treatment / exposure:

Two tissues were used to assess viability after 3-minute exposure (at room temperature), and two were used to assess viability after 60-minute exposure (standard culture conditions).

Duration of post-treatment incubation (if applicable):

After exposure, tissues were rinsed and added to the appropriate MTT reagent solution wells. The plates were then incubated at standard culture conditions for 3 ± 0.1. After incubation the tissues were cleared of excess liquid and placed in wells with isopropanol where they were covered with parafilm and stored refrigerated at 2-8ºC until the last exposure time was harvested. The plates were then shaken for 2-3 hours at room temperature.

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure (Test item)

Value:

103.2

Negative controls validity:

valid

Remarks:

Sterile, deionized water

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure (test item)

Value:

100.6

Negative controls validity:

valid

Remarks:

Sterile, deionized water

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure (Positive control)

Value:

16.4

Positive controls validity:

valid

Remarks:

8N KOH

Remarks on result:

positive indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure (Positive control)

Value:

16.4

Positive controls validity:

valid

Remarks:

8N KOH

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: The test article was not observed to directly reduce MTT in the absence of viable cells.
- Colour interference with MTT: The test article, 5-Octyl-2-Norbornene, was not considered to cause photometric MTT interference.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the assay was accepted if: the positive control resulted in a corrosive classification (i.e., <50% cell viability compared to negative controls, after a 3-minute exposure and/or <15% cell viability compared to negative controls after a 60-minute exposure); and if the mean OD550 value of the negative control tissues was ≥ 0.8 and < 2.8.

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, within acceptable parameters

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item, 5-Octyl-2-Norbornene, met the criteria to be classified as non-corrosive.