Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August 2017 - 04 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
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)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Physical appearance: colourless to pale yellow liquid
Storage conditions: at room temperature protected from light, container flushed with nitrogen.
Specific details on test material used for the study:
No correction factor for purity required.

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
TEST SYSTEM:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified 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.
- Justification: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 μL
- The test material was directly applied to the tissues which were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS.

POSITIVE CONTROL
- Amount applied: 50 μL Methyl Acetate

NEGATIVE CONTROL
- Amount applied: 50 μL Milli-Q water
Duration of treatment / exposure:
30 minutes ± 2 minutes
Duration of post- treatment incubation (in vitro):
Post-soak: 12 ± 2 minutes; incubation after the post-soak: 120 minutes ± 15 minutes
Number of animals or in vitro replicates:
2 tissues per test item and 2 tissues for the negative and the positive control each.
Details on study design:
- Details of the test procedure used:
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23491)
- Duration and temperature:
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS and incubated at standard culture conditions for 30 ± 2 minutes.
* exposure: 30 minutes ± 2 minutes at 37.0 ± 1.0°C
After the exposure period, the tissues were thoroughly rinsed with Ca2+Mg2+-free DPBS (brought to room temperature) to remove residual test item.
* post-soak immersion: 12 ± 2 minutes at room temperature in assay medium
* incubation: 120 minutes ± 15 minutes at 37°C in assay medium + 180 ± 10 minutes with MTT

- The test item was checked for possible color interference and possible direct MTT reduction before the study was started.

- Cell viability measurements:
After incubation, the tissues were incubated with 0.3 mL MTT-medium (1.0 mg/ml) for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were transferred to a 6-well plate containing 2 mL isopropanol. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking. 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.

- Evaluation criteria:
* The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
* The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60%.

- Acceptability criteria:
* The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
* The mean relative tissue viability of the positive control should be <50% relative to the negative control.
* The difference between the % tissue viabilities of the two identically treated replicates should be <20%.

Results and discussion

In vitro

Results
Irritation parameter:
other: Mean cell viability (%)
Value:
78
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
Mean tissue viability: 36%
Remarks on result:
other: SD: 6.6%
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: see 'any other information on results' for historical data. Results for the positive control were within the historical data range and therefore showing that the test system was functioning properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was between >0.8 and <2.5 (i.e., 2.005)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., 36%)
- The difference between the % tissue viabilities of the two identically treated replicates was <20% (i.e., 2.9, 6.6 and 2.1 for the negative control, the treatment group and the positive control respectively)

Any other information on results incl. tables

Table 1 Historical data for EpiOcular studies

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

1.077 – 1.805

0.029 – 0.793

2.11 – 48.25

Mean

1.52

0.42

26.86

SD

0.21

0.23

14.11

n

16

16

16

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2013 to May 2017.

Table 2 Individual OD570 measurements

 

A

(OD570)

B

(OD570)

Negative control

OD570 measurement 1

OD570 measurement 2

 

2.0133

1.9993

 

2.0902

2.0852

FLORANTONE T

OD570 measurement 1

OD570 measurement 2

 

1.7194

1.6748

 

1.5154

1.5050

Positive control

OD570 measurement 1

OD570 measurement 2

 

0.7296

0.7209

 

0.7955

0.7726

OD = Optical density

Duplicate exposures are indicated by A and B.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008
Conclusions:
In an in vitro skin corrosion EpiOcular test, Florantone T was not irritant or corrosive to the eyes with a mean cell viability of 78% compared to the negative control. Based on these results, the test item is not classified according to GHS and according to Regulation (EC) No. 1272/2008.