Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 - 12 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.

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 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:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized
Test material form:
liquid: viscous
Details on test material:
Appearance: Off white paste

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Duration of treatment / exposure:
30 ± 2 mins
Duration of post- treatment incubation (in vitro):
120 ± 15 minutes at 37 ºC
Number of animals or in vitro replicates:
2 tissues per test group
Details on study design:
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23438 kit A) (MatTek Corporation, Ashland MA, U.S.A)

The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

On the day of receipt, the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 ml fresh pre-warmed Assay Medium, which was refreshed after approximately 1 hour. Assay Medium was supplied by MatTek Corporation, Ashland, USA.

All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 - 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

The test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the tissues during the exposure. To assess the colour interference, 50 µl of the test item or 50 µl sterile Milli-Q water as a negative control was added to 1.0 ml Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, 50 µl of the test item or 50 µl sterile Milli-Q water as a negative control was added to 2.0 ml isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.

The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 µl of the test item was added to 1 ml MTT 1 mg/ml MTT solution. The mixture was incubated for approximately 3 hours at
37.0 ± 1.0°C in the dark. A negative control, 50 µl sterile Milli-Q water was tested concurrently. If the MTT solution colour turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

The test was performed on a total of 2 tissues per test item together with a negative control and positive control. Before the assay was started the entire tissues was pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.

Two tissues were treated with 50 µl MilliQ water (negative control) and 2 tissues with 50 µl Methyl Acetate (positive control) respectively. 50 µl of the undiluted test item was added into the 6-well plates on top of the tissues. After the exposure period with the test item (30 ± 2 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for a 12 ± 2 minutes immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 ml MTT-medium
(1.0 mg/ml). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 ml of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. Formazan was extracted with 2 ml isopropanol refrigerated for 18 ± 2 hours in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

A test item is considered to be positive in the in vitro eye irritation test if:

The relative mean tissue viability of two individual tissues after exposure to the test item is ≤ 60% of the mean viability of the negative controls, requiring classification for eye irritation or serious eye damage (UN GHS Category 1 or 2).

A test item is considered to be negative in the in vitro eye irritation test if:

The relative mean tissue viability of two individual tissues after exposure to the test item is > 60% of the mean viability of the negative controls, requiring no classification for eye irritation or serious eye damage (UN GHS No Category).

Results and discussion

In vitro

Results
Irritation parameter:
other: cell viability (%)
Run / experiment:
Undiluted test item
Value:
97
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: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative tissue viability of the positive control should be <50% relative to the negative control.
- Range of historical values if different from the ones specified in the test guideline: N/A

Any other information on results incl. tables

Table 1: Mean adsorption values

 

A

(OD570)

B

(OD570)

Mean

(OD570)

 

SD

Negative control

1.713

1.842

1.778

±

0.091

Test item

1.509

1.927

1.718

±

0.296

Positive control

0.589

0.588

0.589

±

0.001

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability

 

Mean tissue viability (percentage of control)

Standard deviation
 (percentage)

Negative control

100

5.1

Test item

97

17

Positive control

33

0.03

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is non-irritant in the EpiOcular™ test under the experimental conditions previously described.
Executive summary:

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 33% after 30±2 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within 0.5 and 2.8. The standard deviation value of the percentage viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30±2 minutes treatment with the test item compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test item was above 60% after 30 ± 2 minutes treatment the test item is considered to be non-irritant.

Finally, it is concluded that this test is valid and that the test item is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.