Dialkyl C18 and C18-unsaturated phosphonates

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25/09/2017 - 08/10/2017

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dialkyl C18 and C18-unsaturated phosphonates

EC Number:

701-298-1

Cas Number:

64051-29-2

Molecular formula:

Not applicable for a UVCB Substance

IUPAC Name:

Dialkyl C18 and C18-unsaturated phosphonates

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Alkenyl phosphonate
- Expiration date of the lot/batch: 30 June 2018
- Purity test date: 08/06/2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature
- Stability under test conditions: yes

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Justification for test system used:

This model is a three-dimensional human epidermis model, which consists of adult humanderived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes are cultured for 13-days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22.5 hours at 37°C.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed.
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1- Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
2- Test for Color Interference by the Test Item:
Alkenyl phosphonate 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 Alkenyl phosphonate 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.
3- Test for Reduction of MTT by the Test Item
Alkenyl phosphonate was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 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, 25 µl sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement):
* The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) 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 and the Standard Deviation value (SD) of the % viability should be =<18.
b) The mean relative tissue viability of the positive control should be =< 50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be =< 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be =< 18.

* A test item is considered irritant in the skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL

VEHICLE: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL PBS.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL 5% SDS.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 +/- 0.5 minutes treatment with Alkenyl phosphonate

Value:

103

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:

- OTHER EFFECTS:
- Colour interference with MTT: Alkenyl phosphonate was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that Alkenyl phosphonate did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions of this study, the relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Alkenyl phosphonate compared to the negative control tissues was 103%. Since the mean relative tissue viability for Alkenyl phosphonate was above 50% Alkenyl phosphonate is considered to be non-irritant. Therefore, no classification is required according to EU criteria.

Executive summary:

In a skin irritation study (OECD 439, Kr.1, GLP), 25 µL of undiluted Alkenyl phosphonate was applied directly on top of the skin tissue for 15 ± 0.5 minutes (EPISKIN Small model (EPISKIN-SM^TM)).

After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item. 

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Alkenyl phosphonate compared to the negative control tissues was 103%. Since the mean relative tissue viability for Alkenyl phosphonate was above 50% after 15 ± 0.5 minutes treatment Alkenyl phosphonate is considered to be non-irritant.

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 6%, indicating that the test system functioned properly.

In conclusion, Alkenyl phosphonate is non-irritant in the in vitro skin irritation test under the test conditions of this study. Therefore, no classification is required according to EU criteria.