Trihexyl phosphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 - 30 Nov 2016

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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted July 2015

Deviations:

yes

Remarks:

no demonstration of the technical proficiency in the report, no information provided regarding functionality of test system, no Coefficient of variation provided in the report, acceptability criteria vary from TG OECD 431

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation Service, Bratislava, Slovak Republic

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 material form:

liquid

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200-SCT)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: none specified

Source strain:

not specified

Details on animal used as source of test system:

TEST SKIN MODEL
- Source: MatTek Corporation's Reconstituted Human Epidermal Model EpiDerm™ (Lot No. 23382), In Vitro Life Science Laboratories, Slovak Republic.

TEST METHOD
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous, and granular layers, and a multilayered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlying cell layers, which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTION TO CELL CULTURE CONDITIONS
- EpiDermTM was delivered one day before the pre-incubation of tissues and was stored in the original package at 2-8°C. At Day 1, each culture was removed with sterile forceps from the agarose gel, inspected and transferred to pre-labeled 6-well plates containing 0.9 mL of assay medium per assay well. The EpiDermTM cultures were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 1 hour.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37±1
- CO2 gas concentration (%): 5
- Humidity: maximum

Justification for test system used:

This test guideline addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RhE) obtained from human derived non-transformed epidermal keratinocytes which closely mimics the histological, morphological, biochemical, and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200-SCT)
- Tissue batch number(s): lot # 23382
- Production date: none specified
- Delivery date: day before pre-incubation of tissues
- Date of initiation of testing: 29 Nov 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C in humidified atmosphere of 5 ± 1% CO2 in air during incubation with MTT reagent

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: DPBS (20 times: volume not specified)
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: MRX II (Dynex)
- Wavelength: 540 nm
- Filter: no reference filter was used
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- no information given in report

NUMBER OF REPLICATE TISSUES:
- each treatment was conducted in duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- none were needed as there was no direct reduction of MTT, or test substance colour change

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
- a single experiment was conducted

CELL VIABILITY MEASUREMENTS
- For determining alterations in cell viability, MTT reduction assays were performed. Therefore, the tissues were incubated in 300 µL prewarmed MTT solution for 3 h at 37 ± 1°C and 5% CO2. After aspiration of the MTT solution, tissues were blotted on absorbent paper. Extraction of the formazan product was carried out in 2 mL isopropanol overnight without shaking at room temperature. Each extraction solution in a volume of 200 µL was transferred to a 96-well plate, and absorbances were recorded.

PREDICTION MODEL / DECISION CRITERIA
please refer to Table 1 in "Any other information on materials and methods incl. tables"

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration: undiluted

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 100% purified H2O

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8N

Duration of treatment / exposure:

3 and 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

of negative control

Run / experiment:

test substance, 3 min

Value:

ca. 95.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

of negative control

Run / experiment:

test substance, 1 h

Value:

ca. 97.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

other: mean OD

Run / experiment:

test substance, 3 min

Value:

ca. 1.595

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

other: mean OD

Run / experiment:

test substance, 1 h

Value:

ca. 1.522

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct MTT reduction: no direct MTT reduction by the test substance
- Colour interference with MTT: no coloring potential interference by the test substance

DEMONSTRATION OF TECHNICAL PROFICIENCY: not provided in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, as the OD was ≥ 0.8 (1.678 and 1.566 after 3 min and 1 h, respectively)
- Acceptance criteria met for positive control: yes, as the viability was < 15% (7.4% after 1 h)

Any other information on results incl. tables

Table 2: Raw Blank-corrected data after 3 min exposure to negative and positive controls and test substance

	Blank-corrected data			Mean OD	Viability (%)
Negative control (H2O)	1.703	1.633	1.630	1.656	98.7
	1.738	1.681	1.680	1.700	101.3
Positive control (8N KOH)	0.329	0.313	0.318	0.320	19.1
	0.278	0.274	0.276	0.276	16.5
Test substance	1.667	1.607	1.637	1.637	97.6
	1.576	1.540	1.542	1.553	92.6

Table 3: Raw Blank-corrected data after 1 h exposure to negative and positive controls and test substance

	Blank-corrected data			Mean OD	Viability (%)
Negative control (H2O)	1.587	1.541	1.537	1.555	99.3
	1.653	1.532	1.545	1.577	100.7
Positive control (8N KOH)	0.117	0.119	0.115	0.117	7.5
	0.113	0.112	0.113	0.113	7.2
Test substance	1.498	1.461	1.430	1.463	93.4
	1.629	1.546	1.568	1.581	101.0

Table 4: Historical data (from 06/2010 to 10/2013)

Parameter	Negative control (H2O) [optical density]		Positive control (8N KOH) [% viability]
	3 min	1 h	3 min	1 h
Mean	1.565	1.440	18.85	11.03
SD	0.07	0.04	3.54	0.92
Range	1.456-1.647	1.440-1.817	12.7-22.7	9.8-12.5
Results in this study (600361910)	1.678	1.566	17.8	7.4

The results obtained for the negative and positive controls are slightly out of the historical control data range for the 3 min exposure and the 1 h exposure, respectively, but they 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: non-corrosive (Skin Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)

Conclusions:

Under the conditions of the test, the test substance was shown to have no corrosive potential towards reconstructed human epidermis tissue in the EpiDerm™ model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.