Trihexyl phosphate

Eye irritation

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

Test material information

Constituent 1

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

Results

Results 1

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

Results 2

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