Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Remarks:
Applicant assessment of the data indicates that since no irritation response interaction was observed (i.e. including enhanced sensitivity mean viability limit criteria: 65% suggested in OECD TG 492) with the test item in any of the test tissues that the acceptability of the assay was not impacted.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: 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 (Adopted 28 July 2015).
Deviations:
yes
Remarks:
Minor methodology deviations deemed acceptable by expert judgement. Conclusion would acceptable by expert judgement.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 2015; signature: November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid
- Storage condition of test material: At room temperature protected from light, container flushed with nitrogen and desiccated
- Other: pale yellow liquid

Test animals / tissue source

Species:
other: human-derived epidermal keratinocyte tissues
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: MatTek Corporation (USA)
- Housing: EpiOcular™ (OCL-200-EIT; lot 19595 kit G) tissues were shipped on medium-supplemented agarose gels in a 24-well plate. On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature for about 15 minutes. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 ml fresh prewarmed Assay medium. Assay medium was supplied by the manufacturer.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Prewarming: All incubations, with the exception of the last 25 minutes of test item incubation at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 56 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 37.0°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 the appropriate responses of reference controls these deviations are considered not to affect the study integrity.
Killed tissues (Lot: 19580 kit A, 19581 kit I and 19582 kit G): Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them at room temperature. Further use of killed tissues was similar to living tissues.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: TWO tissues were treated with 50 μl MilliQ water (negative control) and TWO tissues with 50 μl Methyl Acetate (positive control) respectively.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
30 ±2 minutes
Observation period (in vivo):
After treatment the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed.
Number of animals or in vitro replicates:
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After pre-incubation of EpiOcular™ tissues and after completion of the treatment with 20 uL PBS, the negative and positive control and the test item was added into the insert atop the concerning EpiOcular duplicate tissues. The plates were placed into the incubator for 24 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2 and room temperature for 7 minutes (this deviation was not considered to impact the study integrity due to appropriate Positive Control and Negative Control responses). 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 to remove residual test item. The test item was difficult to remove from the inserts, with the result that some test item precipitation stuck to the edges of the tissue and the inserts. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (37°C) (the post-soak medium was brought to 37°C instead of room temperature; this deviation was considered to be acceptable since this is the normal culture conditions for the cells and post-soak procedure was performed at room temperature; additionally appropriate control responses were observed) in a pre-labeled 12-well plate for a 12 ± 2 minute 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 with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 ml isopropanol refrigerated for 18 ± 2 hours in the dark. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Plate Reader.

SCORING SYSTEM:
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of formazan formed, as monitored by the absorbance. The results for the time-dependant cytotoxicity curves can be presented as the arithmetic mean ± standard deviation. By setting the absorption of the negative control to 100% cell survival the relative absorption calculated for the test item and the positive control can also be described as tissue viability. Appropriate freeze dried tissue samples and/or correction for colour interference is also conducted.

TOOL USED TO ASSESS SCORE: OD was read in a plate spectrophotometer at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue.

Results and discussion

In vitro

Results
Irritation parameter:
other: Mean Relative Absorbance (% of negative control)
Value:
90
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: S.D. 0.045 or 28% (n=2); Corrected for colour interference and non-specific reduction of MTT by the test item.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: NEGATIVE CONTROL: Mean Relative Absorbance (% of negative control): 100% with S.D. 0.045 (n=2)
- Acceptance criteria met for positive control: POSITIVE CONTROL (methyl acetate): Mean Relative Absorbance (% of negative control): 48% with S.D. 0.005 (n=2)
- Range of historical values if different from the ones specified in the test guideline: The positive control had a mean cell viability after 30 ± 2 minutes exposure of 48%. The absolute mean OD570 of the negative control tissues was within 0.8 and 2.6. The standard deviation value of the percentage viability of two tissues treated identically was less than 3% for the negative and positive controls, indicating that the test system functioned properly.

In vivo

Other effects:
No pH effect of the test substance was observed on the rinsing medium.

Any other information on results incl. tables

Table 1. Mean absorption in the EpiOcular™ test with the test item

 

A

(OD570)

B

(OD570)

Mean

(OD570)

 

SD

Negative control

1.674

1.611

1.643

±

0.045

Test item#1

1.809

1.163

1.486

±

0.457

Positive control

0.785

0.793

0.789

±

0.005

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0423). Isopropanol was used to measure the background absorption.

#1 Corrected for colour interference and non-specific reduction of MTT by the test item.

 

Table 2. Mean tissue viability in the EpiOcular™ test with the test item

 

Mean tissue viability (percentage of control)

Negative control

100

Test item

90

Positive control

48

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test substance is not a eye irritant or corrosive.
Executive summary:

The study was performed according to OECD TG 492 to assess the irritancy potential of the test material to the eye means of the Human Cornea Model Test using the EpiOcular™ model in accordance with GLP. The test item was tested through 50 μl topical application for 30 minutes of tissues of the human cornea model EpiOcular™. The methyl acetate positive control had a mean cell viability of 48% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within 0.8 and 2.5. Thestandard deviation value of the percentage viability of two tissues treated identically was less than 3% for the negative and positive control, indicating that the test system functioned properly. The test item interacted with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) therefore in addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific reduction of MTT by the test item was 0.9% of the negative control tissues. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues. The test item showed colour interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test item was 0.2% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay. Since the test item both interacted with MTT and showed colour interference, in addition to the normal procedure, two freeze-killed tissues were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour in killed tissues was 0.3% of the negative control tissues. The OD of the freeze-killed treated tissues without MTT assay was added to the ODs of the test item treated viable tissues with MTT assay to avoid double correction. 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 90%. The standard deviation of two tissues treated with the test item was 28% which is above the acceptance criterion of 20%. Since both individual viabilities were clearly negative (110 and 71%), this was judged to not affect the study outcome. Applicant assessment of the data indicates that since no irritation response interaction was observed (i.e. including enhanced sensitivity mean viability limit criteria: 65% suggested in OECD TG 492) with the test item in any of the test tissues that the acceptability of the assay was not impacted. Under the conditions of this study, since 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.