[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/11/2017 - 20/02/2018.

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

EC Number:

262-811-8

EC Name:

[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Cas Number:

61477-96-1

Molecular formula:

C23H27N5O7S

IUPAC Name:

[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Test material form:

solid

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™

Source species:

human

Cell type:

other: adult human-derived epidermal keratinocytes

Cell source:

other: human adult donors, not specified.

Source strain:

other: 00267

Justification for test system used:

The EpiDerm™ model has been validated for irritation testing and its use is recommended by the relevant OECD guideline (OECD No. 439); therefore, it was considered suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis (MatTek In Vitro Life Science Laboratories, Bratislava).
- Tissue batch number(s): 25867
- Production date: 18.12.2017
- Delivery date: 19.12.2017
- Date of initiation of testing: 05.12.2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (15-25ºC).
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 1 mL DPBS, 15 times at 1-min intervals.
- Observable damage in the tissue due to washing: no.
- Modifications to validated SOP: no.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3h
- Spectrophotometer: FLUOstar Omega (BMG Labtech)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: OD range 0.0 - 3.5.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.965 ± 0.215
- Barrier function: 4.9 h
- Morphology: functional startum corneum, viable basal cell layer and intermediate spinous and granular layers.
- Contamination: no.
- Reproducibility: yes.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 1 hour exposure is less than 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 1 hour exposure is greater than or equal to 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 ± 2.5 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

1 h ± 0.5 min

Duration of post-treatment incubation (if applicable):

42h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

106.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

OD (mean) = 2.183

Positive controls validity:

valid

Remarks:

viability = 2.2%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Following exposure with the test item, the mean cell viability was 106.1% 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 validity criteria were met.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no, no data correction was necessary.
- Colour interference with MTT: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: This proficiency verification with 10 reference chemicals, in the in vitro skin irritation test, in the EpiDerm™ model, used in OECD 439 (2010) demonstrated that the laboratory is proficient to perform this study. 4 non-irritant chemicals all gave a clearly non-irritant response and the 5 irritant chemicals all gave a clearly irritant response.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD of the negative control tissues must be ≥ 0.8 and ≤ 2.8. The mean OD value was 2.183.
- Acceptance criteria met for positive control: yes, the mean viability of positive control tissues expressed as % of the negative control tissues must be ≤ 20%. The mean viability of positive control tissues was equal to 2.2 %.
- Acceptance criteria met for variability between replicate measurements: yes, standard deviation (SD) within replicates should be ≤ 18%. Standard deviation for tissues treated with negative control, positive control and test item were equal to 11.5 %, 0.1 %, 15 %, respectively.
- Values for negative control and for positive control were within the range of historical data of the test facility.

Any other information on results incl. tables

Table 1. Summary of results.

	Negative control	Positive control	Test item: Piperacillin Acid
Tissue no. 1 viability	85.3	2.1	123.3
Tissue no. 2 viability	108.5	2.3	105.1
Tissue no. 3 viability	106.3	2.1	89.9
Mean Tissue viability	100	2.2	106.1
SD (±)	11.5	0.1	15

The mean OD value for blank was 0.027 ± 0.0004.

Table 2. Blank correlated absorbance values (OD 570 nm).

OD 570 nm		Negative control		Positive control		Test item: Piperacillin Acid
Tissue number	Measurement replicate	OD 570 nm	Mean	OD 570 nm	Mean	OD 570 nm	Mean
1	A	1.873	1.861	0.047	0.046	2.704	2.692
	B	1.849		0.046		2.681
2	A	2.388	2.368	0.051	0.051	2.345	2.294
	B	2.347		0.051		2.243
3	A	2.354	2.32	0.044	0.045	1.989	1.963
	B	2.286		0.046		1.936
Mean of the three tissues		2.183		0.047		2.316

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not irritating according to EU criteria.

Conclusions:

The test item is not irritant to skin.

Executive summary:

An in vitro skin irritation test of the test item was performed in an EpiDerm™ reconstructed human epidermis model, according to OECD TG 439 (GLP study). Disks of EpiDerm™ (three units) were treated with the test item and incubated for 1 hour at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (DPBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2 and 95% RH. 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% CO2 protected from light. The precipitated formazan crystals were then extracted and quantified spectrophotometrically. Concurrent negative and positive controls were run, and all validity criteria were met. The mean cell viability was 106.1% compared to the negative control, while the viability of the positive control was 2.2%. The obtained value is above the threshold of 50%, therefore, the test item was considered as non irritant to the skin.