benzhydryl (6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-trityloxyiminoacetyl]amino]-3-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2017 to 10 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (28 July 2015)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EC) No 761/2009 of 23 July 2009, ANNEX III, B.46., “In Vitro Skin Irritation Reconstructed Human Epidermis Model Test”, amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

diphenylmethyl (6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-3-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

EC Number:

609-462-3

Cas Number:

376653-37-1

Molecular formula:

C44H34N6O6S2

IUPAC Name:

diphenylmethyl (6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-3-formyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Name: BAL0001024
CAS number: 376653-37-1
Batch/Lot number: 013
Appearance: Brown powder
Purity: 63.1% (content by qNMR)
Expiry date: 31 May 2020
Storage conditions: Under inert gas, protected from light and humidity (tight closed container), frozen (≤-15 °C)
Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-045, Expiry Date: 13 November 2017) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere.

Application and rinsing (Day 0)
Test Item
As the test item was solid, 10 µL of distilled water was first applied to the epidermal surface in order to improve contact between test item and epidermis and then 10 mg of the test item was applied, to the epidermal surface, and if necessary spread gently and evenly on to the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

Negative and positive controls
50 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for 15 minutes (± 0.5 min) at room temperature (22.3-24.5°C).
After the 15 minute incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove, as much as possible, any residual material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

MTT test (Day 2)
After the 42 hours incubation, all EPISKINTM (SM) units were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

Formazan extraction (Day 2)
After incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 µL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (Day 2)
Following the formazan extraction, 2×200 µL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 µL/well) was used as blank.
The proper status of the Spectrophotometer was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until
August 2018) at the required wavelength on each day before use.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg of the test item
50 µL of negative control or positive control

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

89.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary and any false estimation of viability can be excluded.
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in the table below.After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean corrected OD value of the three negative control tissues was in the recommended range (0.776). Standard deviation of the viability results for negative control samples was 5.5%.
The positive control treated tissues showed 7.6% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.3%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 5.3 %.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative Control: Phosphate buffered saline	1	0.864	0.817	105.3
	2	0.827	0.780	100.4
	3	0.779	0.732	94.3
	Mean	--	0.776	100.0
Positive Control: 5% (w/v) SDS solution	1	0.106	0.059	7.6
	2	0.108	0.061	7.9
	3	0.103	0.056	7.2
	Mean	--	0.059	7.6
Test Item: BAL0001024	1	0.694	0.647	83.4
	2	0.759	0.712	91.7
	3	0.772	0.725	93.3
	Mean	--	0.695	89.5

Notes:

1. Mean blank value was 0.047

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with BAL0001024, the mean cell viability was 89.5% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
Based on the results of a corrosivity test (performed by Citoxlab Hungary Ltd., study code: 17/220-039B) the test item is non-corrosive.
In conclusion, in this in vitro EPISKINTM (SM) model test with BAL0001024 (Batch number: 012), the results indicate that the test item is non-irritant to skin (No Category).

Executive summary:

An in vitro skin irritation test of BAL0001024 test item was performed in a reconstructed human epidermis model. EPISKIN^TM(SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

 

Disks of EPISKIN^TM(SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure to the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5% CO2 in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5% CO₂in a >95% humidified atmosphere protected from light. The precipitated formazan crystals, the concentration of which indicates cell viability, were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). For each treated tissue, the cell viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

Following exposure with BAL0001024, the mean cell viability was 89.5% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

 

Based on the results of a corrosivity test (performed by Citoxlab Hungary Ltd., study code: 17/220-039B) the test item is non-corrosive.

 

In conclusion, in this in vitro EPISKINTM (SM) model test with BAL0001024 (Batch number: 012), the results indicate that the test item is non-irritant to skin (No Category).