(6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetyl]amino]-3-[(E)-[(3'R)-1'-[(1,1-dimethylethoxy)carbonyl]-2-oxo-[1,3'-bipyrrolidin]-3-ylidene]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid diphenylmethyl ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2017 to 01 September 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:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, No. 431, (29 July 2016) “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl (3E,3'R)-3-{[(6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-2-[(diphenylmethoxy)carbonyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methylidene}-2-oxo-[1,3'-bipyrrolidine]-1'-carboxylate

EC Number:

609-463-9

Cas Number:

376653-41-7

Molecular formula:

C57H54N8O8S2

IUPAC Name:

tert-butyl (3E,3'R)-3-{[(6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-2-[(diphenylmethoxy)carbonyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methylidene}-2-oxo-[1,3'-bipyrrolidine]-1'-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Name: BAL0001026
CAS number: 376653-41-7
Batch/Lot number: 012
Appearance: Off-white powder
Purity: Content by mass balance: 84.9% w/w
Expiry date: 30 June 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:

other: Not applicable

Details on animal used as source of test system:

Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-035, Expiry Date: 04 September 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 corrosivity 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 corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); 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 (Day 0)
The Assay 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, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.3-25.2°C) covered with the plate lids.

Rinsing (Day 0)
After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT test (Day 0)
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere for 3 hours (±15 minutes) while, protected from light.

Formazan extraction (Day 0)
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure 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 overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Cell viability measurements (Day 1)
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 instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, 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:

20 mg of test item was applied
50 µL of physiological saline (negative control)
50 µL of glacial acetic acid (positive control)

Duration of treatment / exposure:

4 hours

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Two replicates were used for the test item. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

92.9

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:

As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined to be 0.008, Non Specific Colour % (NSCliving%) was calculated as 1.0% (see Table below). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not considered necessary in order to exclude the false estimation of viability.

The mean OD value for the test item treated skin samples showed an 92.9% relative viability compared to the negative control.

VALIDITY OF THE TEST
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 OD value of the two negative control tissues was in the recommended range (0.824).
The two positive control treated tissues showed 0.7% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 1.8%.
The difference of viability between the two negative control tissue samples in the MTT assay was 0.9%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated Non Specific Colour % (NSC_living%) of the Additional Control Tissues

Additional control	Optical Density (OD)			NSC% (living)
		Measured	Blank Corrected
Treated with BAL0001026	1	0.052	0.005	1.0
	2	*0.058	*0.011
	Mean	--	0.008

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).

3. *One well was used for counting because another well was excluded from the OD means calculation (negative value).

 

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

Substance	Optical Density (OD)			Viability (% RV)
		Measured	Blank Corrected
Negative Control: Physiological saline (0.9% (w/v) NaCl)	1	0.868	0.821	99.5
	2	0.875	0.828	100.5
	Mean	--	0.824	100.0
Positive Control: Glacial acetic acid	1	0.055	0.008	1.0
	2	0.051	0.004	0.4
	Mean	--	0.006	0.7
Test item: BAL0001024	1	0.801	0.754	91.4
	2	0.825	0.778	94.3
	Mean	--	0.766	92.9

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).

 

HISTORICAL CONTROL DATA

(updated 18 January 2018)

	Negative control (Physiological saline)	Positive control (Glacial acetic acid)
Minimum optical density (OD)	0.59	0.000
Maximum optical density (OD)	1.516	0.051
Mean optical density (OD)	0.864	0.014
Standard deviation (SD)	0.149	0.010
Number of cases	130	130

Note: All optical density (OD) values measured are background corrected values (measured at 570±30 mm)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure of EPISKINTM (SM) to BAL0001026, the mean cell viability was 92.9% compared to the negative control, which is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in the in vitro EPISKIN™(SM) test model, the results indicate that BAL0001026 is non-corrosive to skin.

Executive summary:

An in vitro skin corrosivity test of the test item BAL0001026 was performed in a reconstructed human epidermis model. The model, EPISKIN^TM(SM) is designed to predict and classify the corrosive 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 corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

 

Disks of EPISKIN^TM(SM) were treated with BAL0001026 and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution, to evaluate cytotoxicity. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. Additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

 

Following exposure to BAL0001026, the mean cell viability was 92.9% compared to the negative control, which is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

 

In conclusion, in this in vitro EPISKIN™(SM) test model, the results indicate that BAL0001026 is non-corrosive to skin.