Phosphoric acid, mono- and di-C6-12-(even numbered)-alkyl esters, potassium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 02, 2017 to October 06, 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU B.40 BIS "In Vitro Skin Corrosion: Human Skin Model Test

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, C6-12-alkyl esters, potassium salts

EC Number:

291-900-4

EC Name:

Phosphoric acid, C6-12-alkyl esters, potassium salts

Cas Number:

90506-39-1

Molecular formula:

UVCB

IUPAC Name:

potassium decyl octyl phosphate

Test material form:

solid

Details on test material:

Appearance: White solid
Batch: RE 14-6

Specific details on test material used for the study:

Chemical name (IUPAC), synonym or trade name: Phosphoric acid, mono- and di-C6-12-(even numbered)-alkyl esters, potassium salts
Appearance: White solid
Batch: RE 14-6
Purity/Composition: UVCB
Test substance storage: At room temperature
Stable under storage conditions until: 27 July 2018 (expiry date)

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm Skin Model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Justification for test system used:

The model consists of normal, human-derived epidermal keratinocytes 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts. Recommended test system in international guidelines (OECD and EC)

Vehicle:

unchanged (no vehicle)

Details on test system:

The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 3 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 µL of the undiluted test item was added into the 6-well plates on top of the skin tissues. For the negative and positive controls, 2 tissues were treated with 50 µl Milli-Q water (negative control) and 2 tissues were treated with 50 µl 8N KOH (positive control) for both the 3-minute and 1-hour time point. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

3 minutes and 1 h

Number of replicates:

4 tissues per test susbtance two tissues were used for a 3-minute exposure and two for a 1-h exposure.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: remaining cell viability after exposure to the test item

Run / experiment:

3 minutes

Value:

ca. 88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: remaining cell viability after exposure to the test substance

Run / experiment:

1 h

Value:

ca. 106

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Test substance was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

The mean absorption at 570 nm measured after treatment with the test substance and controls are presented in Table 1 in below sections. The individual OD570 measurements are presented in table 2.

Table 3 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 88% and 106% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.4%.

In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was < 16%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table1: Mean Absorption in thein vitro skin corrosion test with test substance

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.810	2.167	1.988	±	0.253	1.822	1.893	1.857	±	0.050
Test item	1.791	1.696	1.744	±	0.067	2.082	1.842	1.962	±	0.170
Positive control	0.508	0.158	0.333	±	0.247	0.156	0.117	0.137	±	0.028

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0431). Isopropanol was used to measure the background absorption.

Table 2: Individual OD Measurements at 570 nm

	3-minute application (OD570)        A               B		1-hour application (OD570)        A               B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.8603	2.2101	1.8066	1.9555
	1.8617	2.2242	1.9189	1.9235
	1.8367	2.1960	1.8688	1.9290
Test item OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.8565	1.7476	2.1751	1.8878
	1.8369	1.7341	2.1298	1.8969
	1.8095	1.7360	2.0712	1.8701
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.5508	0.2007	0.2011	0.1605
	0.5511	0.2017	0.1988	0.1604
	0.5525	0.2020	0.1984	0.1601

OD = Optical density

Duplicate exposures are indicated by A and B.

Table 3: Mean Tissue viability in the in vitro skin corrosion test with test substance

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	88	106
Positive control	17	7.4

Table 4: Coefficient of variation between tissue replicates

	3 minute	1 hour
Negative control	16	3.8
Test item	5.3	12
Positive control	69	25

CV (%) = 100 - [(lowest OD₅₇₀/highest OD₅₇₀) x 100%]

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Conclusions:

Under the study conditions, the test substance was considered to be non- corrosive to human-derived epidermal keratinocytes (EpiDerm Skin Model).

Executive summary:

A study was conducted to determine the in vitro skin corrosion potential of the test substance, according to OECD Guideline 431and EU Method B.40 BIS: "In VitroSkin Corrosion: Human Skin Model Test (EpiDerm Skin Model)in compliance with GLP. The test substance was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint. Duplicates were exposed to test substance for 3 minutes and 1 hour. Post treatment period, a determination of the cytotoxic effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion was expressed as the remaining cell viability after exposure to the test substance. The positive control had a mean relative tissue viability of 7.4% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤16%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 88% and 106%, respectively.Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive. Under the study conditions, the test substance was considered to be non- corrosive to human-derived epidermal keratinocytes (EpiDerm Skin Model) (Groot, 2017).