5-octylbicyclo[2.2.1]hept-2-ene

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

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: 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.

Test animals / tissue source

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)

Test system

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.

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).