[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 18, 2021 to November 26, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid, (4-morpholinylmethylene)bis-, tetraethyl ester

Cas Number:

59646-46-7

Molecular formula:

C13H29NO7P2

IUPAC Name:

Phosphonic acid, (4-morpholinylmethylene)bis-, tetraethyl ester

Test material form:

liquid: viscous

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of
normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper
parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

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

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

Amount/concentration applied:

30 µl (47 µL/cm²)

Duration of treatment / exposure:

60 min exposure

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Test system

Details on study design:

PRE-EXPERIMENTS
To check the non-specific MTT-reducing capability of the test item 30 μL of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator. The test material sedimented. The mixture did not turn blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were not necessary.
To check the colouring potential of the test item 30 μL of the test item was mixed per 300 μL aqua dest. and/or per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min.
As the test item was classified as non-irritant and colouring was detected by unaided eye-assessment, the light absorption of the chemical in water and in isopropanol in the range of 570 ± 30 nm was measured. Since there was no absorption in the relevant range, the additional test with viable tissues and the quantitative corrections were not necessary.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after
mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of
possible false-negative results were necessary.

EXPERIMENTAL PROCEDURE
Upon receipt of the EpiDermTM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for 60 ± 5 min. Subsequently the tissues were transferred into new wells containing 0.9 mL pre-warmed assay medium per well and were incubated for 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing of all tissues, all plates were incubated for 25 ± 1 min under the sterile flow and for the remaining time of 35 ± 1 min transferred to the incubator.
Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.
The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay
medium per well and the tissue surface was dried using a sterile cotton tip. The plates were postincubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.
After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 μL pre-warmed MTT medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%.
After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature for at least 2 h with gentle shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 min on a plate shaker and the
inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 μL aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

71.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The relative mean
tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the skin irritant potential of the test item was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as nonirritant.
Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.
The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (71.7%) after 60 min treatment and 42 h post-incubation.