Bis(2-ethylhexyl) phosphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Sep - 27 Nov 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SCT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SCT).

Version / remarks:

Version 07 -Nov- 2014

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayer, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) phosphonate

EC Number:

222-904-6

EC Name:

Bis(2-ethylhexyl) phosphonate

Cas Number:

3658-48-8

Molecular formula:

C16H35O3P

IUPAC Name:

bis(2-ethylhexyl) phosphonate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The test was carried out with the reconstituted three-dimensional human skin model EpiDermTM (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDermTM skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (TM): reconstructed three-dimensional human epidermis model
- Tissue Lot number(s): 25840
- Date of initiation of testing: 06 Sep 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with DPBS to remove any residual test item; excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 42 h at 37 ± 1 °C, 5.0% CO2
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC assay. 4 hours, n=3; the OD value (540 – 570 nm) locate in 1.0 – 3.0.
- Barrier function: ET-50 assay. 100 μL 1% Triton X- 100, 4 time – points. N=3, MTT assay; the ET-50 time locate in 4.77 - 8.72.

NUMBER OF REPLICATE TISSUES: 3 each, for the test substance, positive and negative control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTI ON: single experiment.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after 60 minutes exposure and 42 h post-incubation is less or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure and 42 h post-incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 μL test item (undiluted) per tissue (47 μL/cm²)

NEGATIVE CONTROL
- Amount applied: 30 μL DPBS

POSITIVE CONTROL
- Amount applied: 30 μL SDS solution
- Concentration: 5%

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 ± 4 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 ± 1 min

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.
- Colour interference with MTT: The mixture of 30 μL test item per 300 μL aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean absolute OD570 of the three negative control tissues was >= 0.8 and <= 2.8.
- Acceptance criteria met for positive control: the mean relative tissue viability (% negative control) of the three positive control tissues was <= 20% (3.3 %).
- Acceptance criteria met for variability between replicate measurements: the standard deviation (SD) of viability of replicate tissues of all dose groups was <= 18% (0.4% - 4.3%).

Any other information on results incl. tables

Table 1. MTT assay after 60 min exposure

Name	NK			PC			TM
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	2.378 2.329	2.488 2.472	2.632 2.491	0.147 0.155	0.105 0.107	0.108 0.110	0.124 0.120	0.105 0.106	0.120 0.121
OD570 (blank - corrected)	2.335 2.286	2.446 2.429	2.590 2.448	0.105 0.112	0.062 0.065	0.065 0.067	0.082 0.078	0.062 0.063	0.077 0.078
Mean OD570 of the duplicates (blank - corrected)	2.311	2.437	2.519	0.108	0.063	0.066	0.080	0.063	0.078
Total mean OD570 of 3 replicate tissues (blank - corrected)	2.422*			0.079			0.073
SD OD570	0.105			0.025			0.009
Relative tissue viability [%]	95.4	100.6	104.0	4.5	2.6	2.7	3.3	2.6	3.2
Mean relative tissue viability [%]	100.0			3.3**			3.0
SD tissue viability [%]***	4.3			1.0			0.4
CV [% viabilities]	4.3			31.7			12.4

* Blank-corrected mean OD570 nm of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viaability of the three positive control tissues is ≦ 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≦ 18%.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS Category 2 according to Regulation (EC) No 1272/2008

Conclusions:

CLP: Category 2
The test item showed irritant effects. The test item is therefore classified as "irritant" in accordance with CLP/EU GHS "Category 2".