5-octylbicyclo[2.2.1]hept-2-ene

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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

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

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

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

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

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

Interpretation of results:

GHS criteria not met

Conclusions:

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

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 March 2018 to 9 March 2018 (Laboratory phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes

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, protocted from exposure to light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Test item administered undiluted as supplied
- 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

Test system:

human skin model

Remarks:

Reconstructed Human Epidermis Test Method

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Justification for test system used:

The purpose of this study was to evaluate the skin irritation potential of the test article, 5-Octyl-2-Norbornene, supplied by Materia, Inc., in the context of identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/Category 1 and No Category). The protocol was consistent with OECD guideline 439 “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Skin Model
- Tissue batch number: Not specified
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: 30 November 2017
- Date of initiation of testing: 6 March 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Exposure for a 60-minute period. EpiDerm™ tissues were treated in a laminar flow hood then transferred to an incubator at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for 35±1 minutes, the remainder of the 60 minute exposure period.
- Temperature of post-treatment incubation (if applicable): Tissues were rinsed and washed, then placed into an incubator at standard culture conditions for a post-treatment expression incubation of 42±2 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
After 60±1 minutes of test or control article exposure, tissues were rinsed (sterile, CMF-DPBS) by filling and emptying the tissue insert 15 times. A stream of CMF-DPBS was directed onto the tissue surface. For the test and control articles where a mesh was used, the mesh was carefully removed with forceps (if necessary) after the 5th rinse. After removal of the mesh, the rinsing procedure for the tissue continued 10 times. After the 15th rinse, each of the 3 inserts per treatment group (test article, positive and negative control) was completely submerged, gently swirled, and rinse media dumped in a beaker containing approximately 150 mL of CMF-DPBS specifically assigned for each treatment group; this procedure was repeated three times for each insert of each treatment group. Finally, the tissues were rinsed once more on the inside and outside of the tissue insert with sterile CMF-DPBS from the wash bottle, and the excess CMF-DPBS was decanted. The bottoms of the tissue inserts were blotted on sterile paper towels and the inserts transferred to new 6-well plates containing 0.9 mL of fresh warmed (to 37ºC) Maintenance Medium. The tissue surface was carefully blotted with sterile cotton-tipped applicators to remove any excess moisture, and the tissue surface visually observed for residual test article using a dissecting microscope. The tissues were then placed into an incubator at standard culture conditions for a post-treatment expression incubation of 42±2 hours. After an initial 24±1 hours of incubation, the 6-well plates were removed from the incubator and the tissues transferred into new 6-well plates pre filled with 0.9 mL fresh Maintenance Medium warmed to approximately 37ºC. The tissues were then placed back into an incubator at standard culture conditions for an additional 18±1 hours for the remainder of the 42±2 hour post treatment expression incubation.

- 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: Incubated in the dark at standard culture conditions for at least one hour.
- Spectrophotometer: Molecular Devices Vmax plate reader with the AUTOMIX function selected
- Wavelength: 570 nm (OD570)
- Filter: Not specified
- Filter bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The mean viability of the positive control, 5% SDS, was 2.22%. The standard deviation calculated from individual percent tissue viabilities of the
3 identically treated replicates was <18% for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid.
- Barrier function: yers
- Morphology: EpiDerm™ Skin Kit (MatTek Corporation) as provided by the manufacturer.
- Contamination: None
- Reproducibility: within acceptable parameters

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : The test article, 5-Octyl-2-Norbornene, was not observed to directly reduce MTT in the absence of viable cells.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One valid definitive assay

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability after 60 minutes exposure is ≤50%
- The test substance is considered to be non-irritant to skin if the mean tissue viability after 60 minutes exposure is >50%

Control samples:

not required

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 30 µL of test article
- Concentration (if solution): As supplied, without dilution

NEGATIVE CONTROL
- Amount(s) applied (volume): 30 µL of negative control (Sterile, Calcium and Magnesium Free Dulbecco’s Phosphate Buffered Saline (CMF-DPBS))

POSITIVE CONTROL
- Amount(s) applied (volume): 30 µL of positive control
- Concentration (if solution): 5% Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

60-minute exposure period

Duration of post-treatment incubation (if applicable):

42-hour post-exposure expression period

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

One experiment (Negative control)

Value:

100

Negative controls validity:

valid

Remarks:

Sterile, CMF-DPBS

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

One experiment (Test item)

Value:

102.5

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Once experiment (Positive control)

Value:

2.22

Positive controls validity:

valid

Remarks:

5% Sodium Dodecyl Sulfate (SDS)

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 was not determined to be a colorant (was not considered to have potential interference with the MTT measurement).

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the mean OD570 of the negative control, CMF-DPBS, was 2.211. The mean viability of the positive control, 5% SDS, was 2.22%. The standard deviation calculated from individual percent tissue viabilities of the
3 identically treated replicates was < 18% for the positive control and negative control. Since the acceptance criteria were met, the assay was considered valid.


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

Assay Date	IIVS Test Article Number	Sponsor’s Designation	Conc. (w/v)	Mean Viability	Skin Irritation Prediction+
7 March 2018	17AK89	5-Octyl-2-Norbornene	Neat	102.5	Non-Irritant
	Positive Control	SDS	5%	2.22	Irritant

Interpretation of results:

GHS criteria not met

Conclusions:

Based upon the results of this assay, the test article, 5-Octyl-2-Norbornene, was predicted to be non-irritating to skin, and thus would be classified as GHS No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2018 to 19 April 2018 (Experimental start to experimental completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

EpiOcular™ tissues were initially incubated in assay medium for 1hr 23 mins, the protocol stated 1hr. As refeeding replenishes nutrients/growth factors prior to overnight incubation, as refeeding was performed the deviation had no adverse study impact.

GLP compliance:

yes

Remarks:

No GLP certificate included within report

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Materia Inc., USA
- Lot No.of test material: RP367_ONB-D
- Expiration date of the lot/batch: 9 August 2018 (one year from date of manufucature on COA)
- Purity test date: 25 October 2017 (CoA)

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 study
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable, 5-Octyl-2-Norbornene (as supplied) administered to the test system without dilution.
- 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: None, test article, 5-Octyl-2-Norbornene (as supplied) was administered to the test system without dilution.

Species:

human

Details on test animals or tissues and environmental conditions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions)
- Humidity (%): Not specified

Laboratory phase of the study: 6 April 2018 to 19 April 2018 (Experimental start to experimental completion)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 50 μL of the test article, 5-Octyl-2-Norbornene
- Concentration (if solution): Administered as supplied to the test system without dilution.

VEHICLE
- Amount(s) applied (volume with unit): 50 μL of sterile deionized water

Duration of treatment / exposure:

EpiOcular™ tissues were treated in duplicate with the test article, positive control, or negative conrtrol for 30 minutes at standard culture conditions.

Duration of post- treatment incubation (in vitro):

At the end of the 30 minute treatment period, the test or control articles were removed by extensively rinsing the EpiOcular™ tissues. The tissues were then incubated for 120 minutes at Standard Culture Conditions (Post-treatment Incubation).

Number of animals or in vitro replicates:

Duplicate cultures

Details on study design:

- Details of the test procedure used
In accordence with OECD test guideline 492 “Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labeling for eye irritation or serious eye damage. The EpiOcular™ Human Cell Construct (MatTek Corporation) was used to evaluate the ocular irritation potential of the test article, 5-Octyl-2-Norbornene, in the context of identification and classification of ocular irritation of the test article. The ocular irritation potential was evaluated based upon measuring the relative conversion of MTT (3-[4, 5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) in test article-treated tissues after a 30
minute exposure, followed by a 120 minute post-exposure expression period. Ocular irritation potential of the test article was predicted if the relative viability was less than or equal to 60%. If the relative viability was greater than 60%, then the test article was predicted to not require classification or labelling for ocular irritation (GHS No Category). The protocol met the requirements of the OECD test guideline 492. The test article was tested in a valid definitive assay to determine the potential identification and classification of ocular irritation hazard.

- RhCE tissue construct used, including batch number : EpiOcular™ Human Cell Construct model Kit (MatTek Corporation). Batch not specified.

- Doses of test chemical and control substances used : EpiOcular™ tissues were tested in duplicate with 50 μL of the test article, positive control, or negative control.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Prior to test article or control article applications, each tissue surface was moistened with 20 μL of Ca++Mg++-free D-PBS and incubated at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for 30 minutes. After incubation, the EpiOcular™ tissues were tested in duplicate with 50 μL of the test article, positive control, or negative control. The tissues were then placed back into the incubator after dosing, and incubated at standard culture conditions for the remainder of the 30 minute exposure period. At the end of the 30 minute treatment period, the test or control articles were removed by extensively rinsing the EpiOcular™ tissues.

After rinsing, each cell culture insert was immediately transferred to 5 mL of Assay Medium, in a pre-labeled 12 well plate for 12 minutes of immersion incubation (Post-Soak) at room temperature to remove any test article absorbed into the tissue. At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium decanted off, the insert blotted on absorbent material, and then transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm Assay Medium. The tissues were incubated for 120 minutes at Standard Culture Conditions (Post-treatment Incubation).

- Description of any modifications to the test procedure : A protocol deviation that had no impact on study integrity or validity.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : The test article was not observed to reduce MTT directly in the absence of viable cells. therefore a killed control experiment was not performed. The test article was not observed to be a colorant in isopropanol; therefore a colorant control was not performed.

- Number of tissue replicates used per test chemical and controls (positive control, negative control): Duplicate cultures

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : Absorbance measured with a plate reader at the MTT measurement wavelength (550 nm)

- Description of the method used to quantify MTT formazan :
Assessment of Direct Test Article Reduction of MTT:
The test article was added to a 1.0 mg/mL MTT (Sigma) solution in warm Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Fifty microliters of the test article was added to 1 mL of MTT solution, and the mixture incubated in the dark at standard culture conditions for three hours. A negative control, 50 μL of sterile deionized water, was tested concurrently. If the MTT solution color turned blue/purple, the test article was presumed to have reduced the MTT. The test article, 5-Octyl-2-Norbornene, was not observed to directly reduce MTT in the absence of viable cells.

Colorant Control Test:
The ability of the test article to interfere with photometric MTT measurement was assessed. Fifty microliters of the test article was added to 2.0 mL of isopropanol in a 6-well plate and the mixture incubated at room temperature for 2-3 hours. After shaking, 200 μL aliquots of the isopropanol solutions and two blank samples of isopropanol were transferred to a 96-well plate, and the absorbance measured with a plate reader at the MTT measurement wavelength (550 nm). The absorbance of test article samples was determined by subtracting the mean isopropanol blank value from the absorbance of the test article samples. If the OD550 of the test
article sample was > 0.08, the material has to be considered as possibly interacting with the MTT measurement. The test article had a corrected OD550 value of < 0.08, and was not considered to have photometric MTT interference.

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable) Absorbance measured with a Molecular Devices Vmax plate reader at the MTT measurement wavelength (550 nm)

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Evaluation of Test Results:
The protocol meets the requirements of the OECD test guideline “Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage” (TG 492), and utilizes a prediction model to determine the ocular irritation classification as follows:
If the test article-treated tissue viability is > 60% relative to negative control-treated tissue viability, the test article is identified as not requiring classification and labelling according to UN GHS (No Category).

If the test article-treated tissue viability is  60% relative to negative control-treated tissue viability, the test article is identified as potentially requiring classification and labelling according to UN GHS (Category 1or 2).

In Vitro Result In Vivo Prediction

mean tissue viability ≤ 60% Irritant (Category 1 or 2)
mean tissue viability > 60% No Category

Criteria for a Valid Test
The assay results were accepted when the corrected mean OD550 value for the negative control exposure time was >0.8 and <2.5; and the mean relative viability of the positive control was ≤50%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria . Data not referenced against historical data

- Complete supporting information for the specific RhCE tissue construct used: EpiOcular™ Human Cell Construct Model (MatTek Corporation)

- Reference to historical data of the RhCE tissue construct : Data not referenced against historical data

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals . The test facility has extensive experience handling challenging or novel physical forms and chemistries, and is proficient in the use of this assay

- Positive and negative control means and acceptance ranges based on historical data. No reference to historical control data

- Acceptable variability between tissue replicates for positive and negative controls : Variability considered acceptable.

- Acceptable variability between tissue replicates for the test chemical: Variability considered acceptable.

Irritation parameter:

other: Relative viability (%)

Run / experiment:

One valid definitive assay to determine the potential identification and classification of ocular irritation hazard.

Value:

94.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test facility has extensive experience handling challenging or novel physical forms and chemistries, and is proficient in the use of this assay

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay was accepted if the negative control OD was >0.8 and <2.5
- Acceptance criteria met for positive control: The assay was accepted if the mean relative viability was ≥50%

- Range of historical values if different from the ones specified in the test guideline: No historical data ranges specified for this assay

Mean of OD (550)  SD of OD (550)  Mean of viabilities (%)  SD of viabilities  CV %  Negative control 1.758  0.065 100.0  3.70  3.70   Positive control 0.538 0.051 30.6  2.88  9.40  Test article  1.654 0.033 94.1 1.87 1.99

Interpretation of results:

GHS criteria not met

Conclusions:

The test article, 5-Octyl-2-Norbornene, resulted in a relative viability of 94.1%, and is predicted to not require classification or labelling for ocular irritation (GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

This substance will not be classified as it was not irritating to skin or eyes.