Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 - 19 Jan 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:
adopted 28 July 2015
Deviations:
yes
Remarks:
no historical data of positive and negative controls
GLP compliance:
yes (incl. certificate)
Remarks:
GROlJPE lNTERMlNISTERIEL DES PRODUITS CHIMIQUES, 67, rue Barbes, 94201 Ivry-sur-Seine CEDEX, France

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
human
Strain:
other: EpiOcular™

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 50 µL
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
2 h
Number of animals or in vitro replicates:
in duplicates for each treatment and contol group
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-212-ver2.0, supplied by MatTek Corporation), batch No. 23759
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure (37 °C), 12 min post-exposure immersion (room temperature), 2 h post-exposure (37 °C)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary. Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
- Description of the method used to quantify MTT formazan: The absorbance at 570 nm of each well was measured with a microplate reader (model ELx800, BioTek) and the software Gen5 ELISA V1.05.11 (BioTek). No reference wavelength measurement was used.
- Acceptability criteria: 1) Negative control: OD values of the two replicates in the range> 0.8 and< 2.5; 2) Tissues treated with the positive control substance should show a mean tissue viability < 50%; 3) The difference of viability between two tissue replicates should be less than 20% (or the SD between three replicates should not exceed 18%).
- Decision criteria/Prediction model: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is identified as not requiring classification and labelling for skin irritation. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is identified as potentially requring classification and labelling for skin irritation.

Results and discussion

In vitro

Results
Irritation parameter:
other: % mean relative absorbance of 2 tissues
Run / experiment:
30 min exposure
Value:
100.32
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control: The negative control mean OD values (0.684 and 0.585) were not in the range of > 0.8 and < 2.5. Considering the results obtained, this deviation is considered to have no impact on the conclusion of the study.
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (5.04%).
- Deviations from acceptability criteria: The difference of viability between 2 tissues for the test item was 34.52%, instead of 20% as initially scheduled. Considering the results obtained, this deviation is considered to have no impact on the conclusion of the study.

Any other information on results incl. tables

Table 1: Results after 30 min incubation time

 

 

 

Tissue

 

OD

 

Mean OD/ disc (#)

 

Mean OD/ product

Viability

%

Meanviability

%

Differenceofviability

%

 

Conclusion

 

 

0.758

 

 

 

 

 

 

 

I

0.635

0.684

 

107.80

 

 

Negativecontrol

 

0.659

 

0.635

 

100.00

15.60

 

0.558

 

 

 

2

0.607

0.585

 

92.20

 

 

 

 

0.590

 

 

 

 

 

 

 

0.032

 

 

 

 

 

 

 

3

0.032

0.032

 

5.04

 

 

 

Positivecontrol

 

0.033

 

0.032

 

5.04

0.00

UN GHS Category 2 or I

 

0.039

 

 

 

4

0.029

0.032

 

5.04

 

 

 

 

 

0.029

 

 

 

 

 

 

 

 

0.622

 

 

 

 

 

 

 

5

0.811

0.746

 

117.57

 

 

 

Test item

 

0.805

 

0.637

 

100.32

34.52

No Category

 

0.519

 

 

 

6

0.517

0.527

 

83.06

 

 

 

 

 

0.545

 

 

 

 

 

 

Note: the optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

viab > 60% = No Category

viab </= 60% = Irritant

 

#: :mean of 3 values (triplicate of the same extract)

OD: opticaldensity

Acceptability criteria:

- Negative control: OD values of the two replicates in the range > 0.8 and < 2.5;

- Tissues treated with the positive control substance should show a mean tissue viability < 50%;

- The difference of viability between two tissue replicates should be less than 20% (or the SD between three replicates should not exceed 18%).

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
Under the conditions of the conducted test, the test substance is non irritating towards human-derived epidermal keratinocytes in the EpiOcular™ model.