Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 - 17 Aug 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
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 in 2017
Deviations:
yes
Remarks:
no demonstration of the technical proficiency in the report, acceptability criteria vary from TG OECD 492
GLP compliance:
yes (incl. QA statement)
Remarks:
Slovenska Narodna Acreditacna Sluzba, Bratislava, Slovenska Republica

Test material

Constituent 1
Chemical structure
Reference substance name:
Trihexyl phosphate
EC Number:
219-774-8
EC Name:
Trihexyl phosphate
Cas Number:
2528-39-4
Molecular formula:
C18H39O4P
IUPAC Name:
trihexyl phosphate

Test animals / tissue source

Species:
human
Strain:
other: Keratinocyte strain: 4F1188
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. The ability to expose the tissue topically is essential to model the same kind of progressive injury expected in vivo. It also allows both solid and liquid test materials to be applied directly to the tissue. Relative tissue viability is determinedagainst the negative control-treated constructs by the reduction of the vital dye (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) (MTT). Based on the depth of injury model, the EpiOcular EIT is intended to differentiate those materials that are non-irritants (would not require a warning label in the European chemical classification systems) from those that would require labelling as either GHS 1 or 2.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. All biological components of the used tissue and the kit culture medium have been tested and are free of the presence of viruses, bacteria and mycoplasma. Analysis for tissue functionality and quality was performed and passed.

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
- Concentration: undiluted
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
12 min (Post-soak immersion incubation)
120 min (Post-treatment incubation)
Number of animals or in vitro replicates:
2 replicates
Details on study design:
- Details of the test procedure used
Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye conversion by the EpiOcular™ tissue construct after topical exposure to the test substance. The percent reduction of cell viability in comparison to untreated negative controls is used to predict eye irritation potential.

- RhCE tissue construct used, including batch number
The EpiOcularTM human cell construct (e.g. OCL-200 MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 27000)

- Doses of test chemical and control substances used
50 µL test item; 50 μL positive control (methyl acetate) and 50 μL negative control (destilled water)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
exposure: 30 min at 37 ± 1 °C
post-exposure immersion (post-soak): 12 min at room temperature
post-exposure incubation: 120 min at 37 ± 1 °C

- Number of tissue replicates used per test chemical and controls
2 replicates each

- Wavelength used for quantifying MTT formazan
540 nm

- Description of the method used to quantify MTT formazan
Inserts were removed from the 24-well plate after the incubation time in MTT solution (3 h). After incubation, the cultures were blotted on absorbent paper and transferred to a new 24-well plate and extracted in 2 mL of isopropanol overnight without shaking at 2 - 8°C. 2x 200 μL of each extraction solution were transferred to a 96-well plate and the absorbances (ODs) were recorded in order to determine the cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test substance is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, no prediction can be made (further testing is required)

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
please refer to Table 2 in "Any other information on results incl. tables"

- Complete supporting information for the specific RhCE tissue construct used
A copy of the certificate of analysis provided by the manufacturer is attached to the study report.

- Reference to historical data of the RhCE tissue construct
Tissue viability: OD = 1.295 ± 0.075 (acceptance criteria: 1.1 - 3.0)
Barrier function: ET50 = 28.72 min (acceptance criteria: 12.2 - 37.5)
Sterility: no contamination

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
no information in the study report

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: mean viability (%)
Run / experiment:
test substance
Value:
104.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: mean viability (%)
Run / experiment:
positive control
Value:
40.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.745) of the two negative control tissues lies between 1 and 2.6.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (40.6%) lies below 60% of the control viability.
- Acceptable variability between tissue replicates for positive and negative controls: Yes, as the SD of viabilites for positive and negative controls was 1.75% and 0.03%, respectively, and thus < 20%.
- Acceptable variability between tissue replicates for the test chemical: Yes, as the SD of viabilites for the test chemical was 0.92%, and thus < 20%.

Any other information on results incl. tables

Table 1: MTT results

 

Negative control

Positive control

Test substance

Tissue No.

1

2

1

2

1

2

OD540

(blank-corrected)

1.785

1.765

0.710

0.731

1.835

1.830

1.706

1.727

0.676

0.716

1.793

1.830

Mean OD540

1.745

1.746

0.693

0.723

1.814

1.830

Total mean

OD540 ± SD

1.745 ± 0.000

0.708 ± 0.031

1.822 ± 0.016

Relative tissue

viability [%]

100.0

100.0

39.7

41.4

103.9

104.8

Mean relative tissue
viability ± SD [%]

100.0 ± 0.03

40.6 ± 1.75

104.4 ± 0.92

Table 2: Historical control values (from 08/2010 to 07/2017)

Parameter  Negative control (H20) [optical density] Positive control
(methyl acetate)
[% of viability]
Mean  1.371 19
SD 0.253 6.19
Range   1.058 – 1.566 12.1 – 24.1
Results of Study No. 600395330  1.745 40.6

The results obtained for the negative and positive controls are above the historical control data, but both lie in the range of the acceptance criteria (please refer to "Any other information on materials and methods incl. tables").

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:
CLP: not classified