Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April - 14 April 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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

EC Number:

816-314-3

Cas Number:

927881-65-0

Molecular formula:

C83H87N3O21

IUPAC Name:

Methyl (1R,3S,4R,5R)-3-{[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)carbonyl]amino}-5-({2,4,6-tri-O-benzoyl-3-O-[(2S)-1-(benzyloxy)-3-cyclohexyl-1-oxopropan-2-yl]-β-Dgalactopyranosyl}oxy)-4-[(2,3,4-tri-O-benzyl-6-deoxy-α-L-galactopyranosyl)oxy]cyclohexanecarboxylate

Test material form:

solid: particulate/powder

Details on test material:

White solid
Test item storage: At room temperature

Specific details on test material used for the study:

White solid
Test item storage: At room temperature
Batch: 902/6460031/D/13/1 (is same batch as batch E010017351 on Certificate of Analysis)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: EpiDerm Skin Model (EPI-200, Lot no.: 25761 kit U and V). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

Details on animal used as source of test system:

EpiDerm Skin Model (EPI-200, Lot no.: 25761 kit U and V)

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

water

Details on test system:

EpiDerm Skin Model (EPI-200, Lot no.: 25761 kit U and V).
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.
Test System Set Up:
Tissues:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM.
DMEM (Dulbecco’s Modified Eagle’s Medium):
Supplemented DMEM, serum-free supplied by MatTek Corporation.
MTT medium:
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
Test Item Preparation:
No correction was made for the purity/composition of the test item. The solid test item was applied directly on top of the skin tissue.
Application/Treatment of the Test Item:
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 2 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. The skin was moistened with 25 μl Milli-Q water to ensure close contact of the test item to the tissue and 28.6 to 38.4 mg of the solid 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 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

Amount/concentration applied:

The skin was moistened with 25 μl Milli-Q water to ensure close contact of the test item to the tissue and 28.6 to 38.4 mg of the solid test item
was added into the 6-well plates on top of the skin tissues.

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

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.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

90

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

PF-06460260 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The positive control had a mean relative tissue viability of 7% 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 ³0.8 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 the test item compared to the negative control tissues was 88% and 90%, repectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

Executive summary:

In conclusion, PF-06460260 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.