Decyl dihydrogen phosphate, potassium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 05 May 2017. Experimental completion date: 09 June 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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UN GHS (published 2003, last (6th) revision 2015).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Decyl dihydrogen phosphate, potassium salt

EC Number:

270-390-7

EC Name:

Decyl dihydrogen phosphate, potassium salt

Cas Number:

68427-32-7

Molecular formula:

C10H23O4P.xK

IUPAC Name:

potassium decyl hydrogen phosphate

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

liquid

Details on test material:

Identification: Decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7)
CAS-No.: 68427-32-7
Batch: 1526886
Purity: 56%
Physical state/Appearance: Pale yellow liquid
Expiry Date: 31 March 2018
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiSkin™ Kit Components Needed for the Assay
EpiSkin™ Kit Lot No.: 17-EKIN-023
1 Sealed 12-well plate Contains 12 inserts with EpiSkin™ tissues on agarose
1 12-well plate For MTT viability assay
1 bottle Assay Medium Basic medium for use in MTT assays
1 bottle EpiSkin™ Maintenance Medium Basic medium for incubations

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The
EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly
differentiated model of the human epidermis. It consists of organized basal, spinous and
granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid
layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface
0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose
gels in a 12-well plate and reached Envigo CRS GmbH on 07 June 2017. On the day of
experiment the pre-incubation phase of the EpiSkin™ tissues started.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-warming of EpiSkin™ Tissues
Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates
containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were
incubated for approximately 3 hours.

Treatment
The negative control, positive control and the test item were added into the insert atop the
concerning EpiSkin™ triplicate tissues for 15 minutes.
After the end of the treatment interval the inserts were immediately removed from the 12-
well plate. The tissues were gently rinsed with PBS to remove any residual test material.
Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting
paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues
were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

IL-1 α Immunoassay
Samples of all treatment groups were taken from the wells. The plates were shaken for
approximately 15 minutes to homogenise the released mediators in the medium before
sampling. At least 1.6 mL medium from each well was taken and was stored in the freezer at
–20 °C. Since the results derived from the MTT assay were not
unclear or borderline, the IL-1 α concentration in the medium was not determined and the
taken samples were discarded after report finalization

MTT Assay
A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the 42 ± 1 hour incubation period was completed for all tissues the cell culture inserts
were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period
(37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues
were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred
into plastic vials. The tissue samples were immersed into extractant solution by gently
pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The
tissue samples were completely covered by isopropanol. The formazan salt was extracted for
about 2.5 hours at room temperature with gentle agitation.
Per tissue sample 2 × 200 μL aliquots of the formazan extract were transferred into a 96-well
flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular
Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 nm filter. Mean values were
calculated from the 2 wells per tissue sample.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL (26.3 μL/cm2) of undiluted test item
10 μL of both negative and positive control

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2

Number of replicates:

triplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

20

Vehicle controls validity:

not applicable

Negative controls validity:

not valid

Remarks:

please see acceptance of results section

Positive controls validity:

not valid

Remarks:

please see acceptance of results section

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test item in water did not lead to a change in colour.Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability,decreased to 20.0% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

Concerning acceptance criteria:
•The mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 1.384 to 1.468).
• The rel. standard deviations between tissues of the same treatment group was ≤ 18% (3.2% (negative control) and 13.1% (test item)). For the positive control, the acceptance criteria were not met (28.7%). Reason: extremely low absorbance values of 0.014, 0.014 and 0.022, where small differences cause high rel. standard deviations.

The OECD guideline 439 and the SkinEthic protocol request only the standard deviation being ≤ 18% as acceptance criteria between tissues of the same treatment group. If the standard deviation is considered (0.3%), the acceptance criteria are met.

This deviation from the acceptance criteria is not considered to have an impact on the outcome of the study.
• The mean relative tissue viability of the positive control was ≤ 40% (1.2%).
• The acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.8 mg/mL).
• Historical Data for the negative control are not available. Reason: Envigo CRS GmbH changed the negative control for the test system “In vitro Skin Irritation Assay using RHE supplied by SkinEthic” from deionized water to PBS. The test is performed at Envigo CRS GmbH a long time prior to former a version of OECD 439 guideline, where deionized water or PBS are suggested as negative control. Originally, SkinEthic requested deionized water to be used as negative control in their protocol.

All of Envigo CRS GmbH’s historical data are based on deionized water. In the current SkinEthic protocol, PBS is suggested as negative control. Therefore, Envigo CRS GmbH decided to change the negative control to be in accordance with the SkinEthic protocol, but the number of performed assays using PBS as negative control is still too low to issue historical data.

Historical Data for the positive control are available (see annex 1). But the determined viability value of 1.2% is not within the historical data (viability range: 3.90% -32.73%). Since the historical data at Envigo CRS GmbH are updated at least once a year, after next update the present positive control viability of 1.2% will be within the historical range. Since neither the OECD guideline nor SkinEthic requests historical data for the positive control, the outcome of the study is not impacted by this.

The exceptions from the acceptance criteria did not have an impact on the outcome of the study.

Any other information on results incl. tables

Test Group	Absor-bance 570 nm Tissue Well 1	Absor-bance 570 nm Tissue Well 2	Mean Absor-bance 570 nm	Mean Absor-bance 570 nm*	Mean Absor-bance of 3 Tissues	Standard Deviation of Tissue Absorbance	Relative Absorbance [%] Tissue 1, 2 + 3**	Standard Deviation [%]	Rel. Standard Deviation [%]****	Rel. Absorbance [% of Negative Control]***
Blank	0.050	0.048	0.049	0.000
Negative Control	1.480	1.457	1.468	1.420	1.387	0.045	102.4	3.2	3.2	100.0
	1.469	1.438	1.453	1.405			101.3
	1.432	1.337	1.384	1.336			96.3
Positive Control	0.063	0.062	0.062	0.014	0.017	0.005	1.0	0.3	28.7	1.2
	0.061	0.065	0.063	0.014			1.0
	0.076	0.066	0.071	0.022			1.6
Test Item	0.305	0.290	0.298	0.249	0.277	0.036	18.0	2.6	13.1	20.0
	0.314	0.311	0.312	0.264			19.0
	0.368	0.366	0.367	0.318			22.9

*         Mean of two replicate wells after blank correction

**       relative absorbance per tissue [rounded values]: (100 x absorbance_tissue)/(mean absorbance _{negative control})

***     relative absorbance per treatment group [rounded values]: (100 x mean absorbance_{test item})/(mean absorbance_{negative control})

****      relative standard deviation per treatment group [rounded values]: (100 x standard deviation_{tissue absorbance})/(mean absorbance_tissue)

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7) is irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7) by means of the Human Skin Model Test according to OECD TG 439.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 20.0%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Decyl dihydrogen phosphate, potassium salt (CAS# 68427-32-7) is irritant to skin according to UN GHS and EU CLP regulation.