Fatty acids, C18-unsatd., phosphates

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12.07.2017-06.09.2017

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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

9-(dimethoxyphosphoryl)octadecanoic acid 10-(dimethoxyphosphoryl)octadecanoic acid

Molecular formula:

C20H41O5P

IUPAC Name:

9-(dimethoxyphosphoryl)octadecanoic acid 10-(dimethoxyphosphoryl)octadecanoic acid

Constituent 2

Reference substance name:

9,12-bis(dimethoxyphosphoryl)octadecanoic acid 9,13-bis(dimethoxyphosphoryl)octadecanoic acid 10,12-bis(dimethoxyphosphoryl)octadecanoic acid 10,13-bis(dimethoxyphosphoryl)octadecanoic acid

Molecular formula:

C22H46O8P2

IUPAC Name:

9,12-bis(dimethoxyphosphoryl)octadecanoic acid 9,13-bis(dimethoxyphosphoryl)octadecanoic acid 10,12-bis(dimethoxyphosphoryl)octadecanoic acid 10,13-bis(dimethoxyphosphoryl)octadecanoic acid

Constituent 3

Reference substance name:

Oleic acid

EC Number:

204-007-1

EC Name:

Oleic acid

Cas Number:

112-80-1

Molecular formula:

C18H34O2

IUPAC Name:

octadec-9-enoic acid

Constituent 4

Reference substance name:

Linoleic acid

EC Number:

200-470-9

EC Name:

Linoleic acid

Cas Number:

60-33-3

Molecular formula:

C18H32O2

IUPAC Name:

octadeca-9,12-dienoic acid

Constituent 5

Reference substance name:

Dimethyl (2-oxopropyl)phosphonate

EC Number:

224-110-5

EC Name:

Dimethyl (2-oxopropyl)phosphonate

Cas Number:

4202-14-6

Molecular formula:

C5H11O4P

IUPAC Name:

dimethyl (2-oxopropyl)phosphonate

Constituent 6

Reference substance name:

Dimethyl propylphosphonate

EC Number:

242-555-3

EC Name:

Dimethyl propylphosphonate

Cas Number:

18755-43-6

Molecular formula:

C5H13O3P

IUPAC Name:

dimethyl propylphosphonate

Constituent 7

Reference substance name:

Dimethyl methylphosphonate

EC Number:

212-052-3

EC Name:

Dimethyl methylphosphonate

Cas Number:

756-79-6

Molecular formula:

C3H9O3P

IUPAC Name:

dimethyl methylphosphonate

Test material form:

liquid

Specific details on test material used for the study:

IDENTIFICATION OF THE TEST MATERIAL
- Identification: Fatty acids, C18-unsatd., phosphates.
- CAS number: 68604-99-9
- EC number: 271-708-7
- Appearance: Yellow liquid

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 02 November 2017
- Purity test date: 100% UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No specific handling conditions required
- Solubility and stability of the test substance in the solvent/vehicle: no
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not expected

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 25 μl of the undiluted test item was added into 12-well plates on top of the skin tissues.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm Skin Model (EPI-200, Lot no.: 26708 Kit L and M). 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.
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 (see
figure 1). The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0ºC. The
medium was replaced with fresh DMEM just before C18-unsatd., phosphates. 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 C18-unsatd.,
phosphates. and two for a 1-hour exposure. Fifty 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.
4.7.5. Cell Viability Measurement
The DMEM was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at
37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and
formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room
temperature. The amount of extracted formazan was determined spectrophotometrically at
570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5%

Duration of treatment / exposure:

3-minute and 1-hour time points.

Number of replicates:

2

Test system

Amount / concentration applied:

- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5%

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

6.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 μl of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed. No color change were observed.
- Colour interference with MTT: The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, 10 μl of the test item was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed. No color changes were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 6.8%. The absolute mean OD570(optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range.
- Acceptance criteria met for positive control: the positive control had a mean cell viability of 16% after 15 ± 0.5 minutes exposure. The mean relative tissue viability of the positive control should be <=50% relative to the negative control.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 12%. The SD calculated from individual % tissue viabilities of the three identically treated replicates should be <=18.

Any other information on results incl. tables

Table 1. Mean absorption in the in vitro skin corrosion test with C18 -unsatd. phosphates. 3 -minute application

	A(OD570)	B(OD570)	Mean (OD570)	SD
Negative control	1.692	1.836	1.764	+-0.102
Test item	1.464	1.647	1.555	+-0.129
Positive control	0.115	0.135	0.125	+-0.014

SD= standard deviation, Duplicate exposures are indicated by A and B

Table 2. Mean tissue viability in the in vitro skin corrosion test with C18 -usatd. phosphates, 1 -hour application

	A(OD570)	B(OD570)	Mean (OD570)	SD
Negative control	1.702	1.566	1.634	+-0.096
Test item	0.090	0.106	0.098	+-0.011
Positive control	0.188	0.153	0.171	+-0.025

Table 3. Mean tissue vability in the in vitro skin corrosion test with C18 -usatd. phosphates.

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	88	6.0
Positive control	7.1	10

 

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In conclusion, C18-unsatd., phosphates. is corrosive in the in vitro skin corrosion test under the experimental conditions described.

Executive summary:

The objective of this study was to evaluate C18-unsatd., phosphates. for its ability to induce skin corrosion on on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of C18-unsatd., phosphates. was tested through topical application for 3 minutes and 1 hour. The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 0101891886 of C18-unsatd., phosphates. was a yellow liquid. C18-unsatd., phosphates. was applied undiluted (50 μl) was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 10% 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  11%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with C18-unsatd., phosphates. compared to the negative control tissues was 88% and 6.0%, respectively. Because the mean relative tissue viability for C18-unsatd., phosphates. was below 15% after the 1-hour treatment it is considered to be corrosive. Subsequently, after 3 -min exposure the mean tissue viability was above 25%. In conclusion, C18-unsatd., phosphates. is corrosive, category 1B in the in vitro skin corrosion test under the experimental conditions described.