[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

Endpoint summary

Description of key information

Weight of evidence. Based on the available data, the test item should be classified as Skin Sensitizer Category 1, sub-category 1B according to Regulation (EC) No 1272/2008 (CLP). Four studies have been conducted as part of an integrated approach to support the identification of the sensitisation potential of the test item:

- Test method according to OECD 442C, GLP study. The test item showed mean depletion of 1.06% Cysteine, which reflects no or minimal reactivity, therefore a negative prediction of DPRA.

- Test method according to OECD 442D, GLP study. The test item presented negative results in both runs, with a cell viability > 70% and Imax 1.28 and 0.97 for the first and second runs for KeratinoSens™, respectively. Therefore, the test item shows no sensitisation potential.

- Test method according to OECD 442E, GLP study. The test item presented negative results both runs up to 500 μg/mL. Therefore, the test item may be classified as not skin sensitizer.

However, the obtained results were not coherent with the available evidence showing that the analogue substance piperacillin sodium salt can lead to skin sensitisation (delayed contact hypersensitivity) based on human experience (see section 7.10) and on the evidence from chemical structure related substances (penicillins). Therefore, a confirmatory study was performed.

- Test method according to OECD 429, GLP study. Based on the test results (SI = 8.7, 8.2 and 3.0 at doses of 50, 25, and 10% (w/v), respectively, enlarged lymph nodes at 50% w/v and EC3 = 10% w/v), the test item was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. According to Regulation (EC) No 1272/2008 (CLP), the test item should be classified as Category 1, sub-category 1B.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01/02/2018 - 29/03/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

EURL ECVAM DB-ALM Protocol n.º 154

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

TEST SYSTEM
- Cysteine peptide (Ac-RFAACAA-COOH): source JPT Peptide Technologies GmbH, lot no. 111016HS_MHeW1017
- Lysine peptide (Ac-RFAAKAA-COOH): source JPT Peptide Technologies GmbH, lot no. 120514HSDWW1017
- Solvent/vehicle: acetonitrile, based on the results of the preliminary solubility test.
- Preparation of test item stock solution: the test item was dissolved at 100 mM in acetonitrile without sonication.
- Preparation of test item samples for the reactivity with cysteine peptide: 50 µL of test item formulation was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
- Preparation of test item samples for the reactivity with lysine peptide: 250 µL of test item formulation was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

CONTROLS (preparation)
- Positive control: 100 mM cinnamaldehyde (purity 99.1%, Sigma-Aldrich, lot no. MKCB9907).
- Positive control for cysteine peptide: 50 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
- Positive control for lysine peptide: 250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Reference controls: All the reference control samples were prepared in triplicate at the nominal concentration of 0.500 mM of peptide in the solvent specified below.
- Reference control A: acetonitrile (to check calibration curve accuracy)
- Reference Control B: acetonitrile (to check the stability of the peptide over time)
- Reference Control C: acetonitrile (solvent used both for the test item and the positive control)
- Co-elution controls: 100 mM test item in the appropriate buffer.
- Co-elution control (cys p.): 50 µL test item formulation was incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL acetonitrile.
- Co-elution control (lys p.): 250 µL of test item formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

HPLC ANALYSIS
- Equipment: HPLC system with autosampler, UV detector (200 nm), Zorbax SB C18 (100 x 2.1 mm; 3.5 µm) HPLC analytical column.
- Conditions: sample temperature 25ºC, column temperature 30ºC, injection volume: 7 µL(cys) or 3 µL(lys), flow rate 0.35 mL/min, total analysis time 20 min.
- mobile phase A: acetonitrile + 0.085% TFA
- mobile phase B: milli-Q water + 0.1% TFA
- System suitability: calibration linearity: r2 > 0.990 for both peptides; mean peptide concentration of Reference control A = 0.508 (cys) and 0.498 (lys) = 0.5 ± 0.005 mM.
- Analysis sequence: For each peptide, the analytical sequence included at least: one blank sample (peptide dilution buffer), one calibration curve injected at the beginning of the analytical batch, three reference control A samples, the co-elution control sample, three reference control B samples, reference control C samples (replicate 1), positive control sample (replicate 1), and test item study sample (replicate 1).

ACCEPTANCE CRITERIA
- For the positive control, the mean % peptide depletion value must fall within 60.8 - 100.0% (cys) and 40.2 - 69.4 (lys);
- For the positive control, SD (cys) < 14.9% and SD (lys) < 11.6%;
- For the reference controls, CV% of the peak areas for reference controls B, C must be < 15.0%;
- For the reference controls in the analysis sequence, the mean peptide concentration of Reference control C must be 0.5 ± 0.005 mM;
- For the test item replicates, SD (cys) < 14.9% and SD (lys) < 11.6%;

INTERPRETATION OF RESULTS: Cysteine 1:10-only prediction model.
- 0.00 % ≤ mean % depletion ≤ 13.98 % = No or minimal reactivity = Negative DPRA Prediction
- 13.98 % ≤ mean of cysteine and lysine % depletion ≤ 23.09 % = Low reactivity = Positive DPRA Prediction
- 23.09 % ≤ mean of cysteine and lysine % depletion ≤ 98.24 % = Moderate reactivity = Positive DPRA Prediction
- 98.24 % ≤ mean of cysteine and lysine % depletion ≤ 100 % = High reactivity = Positive DPRA Prediction

Positive control results:

The depletion mean of cinnamaldehyde was 51.08 for lysine peptide and 76.99 for cysteine.

Key result

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion (%)

Value:

1.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no or minimal peptide reactivity

Other effects / acceptance of results:

OTHER EFFECTS:
- Appearance of precipitate (if yes, if precipitate was re-solubilised or centrifuged): precipitate was observed in the positive control samples incubated with the cysteine or lysine peptides, these vials were centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Thus, only supernatants were injected into the HPLC/UV system.
- Co-elution: analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with the cysteine peptide. However, the test item co-eluted with the lysine peptide. The % interference of this co elution could not be evaluated since several peaks were observed at the lysine peptide retention time and therefore no peak integration could have been performed. As a result, the peptide reactivity classification of the test item is only based on the cysteine 1:10 prediction model.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the expected DPRA prediction for the 10 proficiency substances was obtained. The resulted cysteine and lysine depletion values fall within the respective reference range for 9 out of the 10 proficiency substances for each peptide (8 out of 10 expected in OECD guideline).

ACCEPTANCE OF RESULTS:
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied.
- Acceptance criteria met for reference controls: yes, the mean peptide concentrations of the reference control C samples was 0.504 mM (cys) and 0.493 mM (lys), within ± 10% of the nominal concentration; and the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile was 1.0% (cys) and 0.7% (lys).
- Acceptance criteria met for positive control: yes , for cysteine peptide, the mean percent depletion value was 93.69% (SD 0.22), within the acceptance range (60.8 - 100%, SD < 14.9%); and for lysine peptide, the mean percent depletion value was 59.85% (SD 0.86), within the acceptance range (40.2 - 69.4, SD < 11.6%).
- Acceptance criteria met for variability between replicate measurements: yes, the maximum SD for the test item replicates was 0.003 < 14.9% for the percent cysteine depletion value, below 15%. Percent lysine peptide depletion value could not be determined due to co-elution.

Table 1. Test item results: Percent peptide depletion.

Sample no.	Cysteine peptide		Lysine peptide		Depletion rate (%) of the test item	Depletion classification
	Peak area(μV/sec)	% depletion	Peak area(μV/sec)	% depletion
1	2684898	1.18	-	-
2	2680963	1.33	-	-
3	2698543	0.68	-	-
Mean	-	1.06	-	-	1.06	No reactivity / minimal reactivity
SD	-	0.34	-	-
CV	-	32.0	-	-

Table 2. Positive control results: percent peptide depletion.

Sample no.	Cysteine peptide		Lysine peptide		Depletion rate (%) of the test item	Depletion classification
	Peak area(μV/sec)	% depletion	Peak area(μV/sec)	% depletion
1	176093	93.52	366445	60.64
2	173530	93.61	372690	59.97
3	164812	93.93	382312	58.93
Mean	-	93.69	-	59.85	76.77	High reactivity
SD	-	0.22	-	0.86
CV	-	0.2	-	1.4

Table 3. Reference control A.

12/03/18 Sample no.	Cysteine peptide			Lysine peptide
	Area (μV/sec)	Concentration (mM)	%Dev	Area (μV/sec)	Concentration (mM)	%Dev
RC A.1	2737214	0.508	1.6	935028	0.496	-0.9
RC A.2	2732757	0.507	1.5	939950	0.498	-0.4
RC A.3	2735744	0.508	1.6	941904	0.499	-0.1
Mean	-	0.508	1.6	-	0.498	-0.5
SD	-	0.000	-	-	0.002	-
%CV	-	0.1	-	-	0.4	-

Table 4. Reference controls B, C.

12/03/18 Sample no.	Cysteine peptide	Lysine peptide
	Area (μV/sec)	Area (μV/sec)
RC B.1	2726083	936771
RC B.2	2716934	938181
RC B.3	2708476	938965
RC B.4	2667294	921984
RC B.5	2667733	932257
RC B.6	2656596	929823
RC C.1	2699563	937539
RC C.2	2725177	933797
RC C.3	2726258	921633
Mean	2699346	932328
SD	28181	6662
%CV	1.0	0.7

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed mean peptide depletion of 0.30% for Lysine, reflecting no or minimal reactivity. Therefore a negative prediction of DPRA.

Executive summary:

A Direct Peptide Reactivity Assay has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item. The method was performed according to OECD OECD 442C, under GLP. The aim of the study is to evaluate the reactivity of the test item in chemico by quantifying the depletion of synthetic heptapeptides containing either lysine or cysteine.

A preliminary solubility study was conducted for the test item, and based on the results, the test item was prepared in acetonitrile. Peptide solutions were incubated with 100 mM test item solution or 100 mM cinnamic aldehyde solution (positive control), at ratios of 1:10 for cysteine and 1:50 for lysine. Reference controls and co-elution controls were run in parallel. After 24h incubation at 25ºC, the residual peptide concentrations were evaluated by HPLC-UV (220 nm). Under test conditions, the test item co-eluted with lysine, and the % interference could not be determined, so the peptide reactivity classification was only based on the cysteine 1:10 prediction model. The percent cysteine peptide depletion for the test item was 1.06, reflecting no or minimal reactivity, and thus a negative DPRA prediction. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. Based on the test results, the test item shows no sensitisation potential.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25/01/2018 - 23/02/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

EURL ECVAM DB-ALM Protocol n.º 155

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light and humidity.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
- Cell line used, storage conditions and source: KeratinoSens™ (Batch D1, Givaudan), exempt of mycoplasm; storage at -80ºC.
- Passage number and level of confluence of cells: Cells were grown using general culture procedures up to 80-90% confluence; the test item was tested in two independent runs using cells from a different passage number.
- Cell counting method used for seeding prior to testing and measures taken to ensure homogeneous cell number distribution: the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 (DMEM with 9.1% FCS without G-418) and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8E04 cells/mL. Then, 125 µL (1E4 cells) per well were added to four 96-well plates taking care to avoid sedimentation of the cells during seeding. The seeded plates were incubated for 24 ± 1 hours at 37ºC, 5% CO2.
- Luminometer used: Varioscan Flash (Thermo Scientific) 96-well plate Luminometer with injectors and optical density reader.
- Number of repititions and replicates: two independent runs were performed, with three replicates each. New formulations were prepared for each run.
- Test item concentrations: On the basis of a preliminary solubility test results, the test item was dissolved in DMSO at 200 mM and then serial dilutions were performed. Both runs were performed using concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.
- Controls: 1% DMSO in culture medium was used as vehicle/negative control; cinnamic aldehyde was used for the positive control; cytotoxicity was measured by MTT reduction.
- Application procedure and exposure time: After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL treatment medium; from the Master plate 4x, a volume of 50 µL was added to each well of the 3 assay plates and 50 µL to the plate for the cytotoxicity evaluation. All plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells, and the plates were then incubated for 48 (± 2) hours at 37ºC, 5% CO2, 90% humidity.
- Description of evaluation and decision criteria used: The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. If the first two runs were not concordant, a third run is performed and the final outcome is that of the two concordant runs.
The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- the EC1.5 value is < 1000 µM (or < 200 µg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).
- Description of study acceptance criteria used: Each run was considered valid if the following criteria were met:
- the positive control results should be positive, thus gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose response with increasing luciferase activity at increasing concentrations for the positive control,
- the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

Positive control results:

Imax = 5.95 (mean); EC1.5 = 12.47 (geometric mean)

Key result

Run / experiment:

other: #1

Parameter:

other: Imax

Value:

1.28

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: #2

Parameter:

other: Imax

Value:

0.97

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: #1 and #2

Parameter:

other: cell viability (%)

Value:

70

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: As no cytotoxicity was observed, IC30 and IC50 were not calculated.

Run / experiment:

other: #1 and #2

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

- no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period in both run,
- no noteworthy decrease in cell viability were noted (i.e. cell viability > 70%), therefore no IC30 or IC50 was calculated in both runs,
- no statistically significant gene-fold induction above the threshold of 1.5 were noted in comparison to the negative control at any tested concentrations. Thus, no EC1.5 was calculated,
- the Imax values were < 1.5 (i.e. 1.28 and 0.97 in the first and the second run, respectively).

OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: recommended substances for demonstrating technical proficiency with the KeratinoSens™ test method were tested to validate the method.

ACCEPTANCE OF RESULTS:
- All acceptance criteria were fulfilled for the positive and negative controls in each run. These runs were therefore considered to be valid.
- Acceptance criteria met for negative control: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% in each repetition.
- Acceptance criteria met for positive control: yes. A positive result was obtained; gene induction was statistically significant above the threshold of 1.5 in at least one of the tested concentrations; EC1.5 = 12.47 µM, value was within two standard deviations of the historical mean; Imax in the three replicate plates for the positive control at 64 µM was between 2 and 8.
- Acceptance criteria met for variability between replicate measurements: yes.
- Range of historical values if different from the ones specified in the test guideline: Imax = 2.7-22.7, EC1.5 = 2.8-14.8 μM (n=32).

Results for the positive control.

Cinnamic aldehyde	4	8	16	32	64	Imax	EC1.5 (μM)
Run 1	1.1	1.2	1.6	2.3	3.9	3.93	14.24
Run 2	1.2	1.4	1.7	2.4	8.0	7.98	10.92
Mean	1.1	1.3	1.7	2.4	6.0	5.95	-
Geometric mean	-	-	-	-	-	-	12.47
SD	0.1	0.1	0.1	0.1	2.9	2.87	2.35

Results for the negative control

Negative control	Reading (mean)	%CV
Run 1	356709	12.97
Run 2	671367	19.39

Results for the test item.

Test item	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000	Imax	EC1.5
Induction Run 1	1.3	1.2	1.1	1.2	1.1	1.1	1.1	1.1	1.1	1.2	0.9	0.9	1.28
Viability (%) Run 1	95	96	103	105	104	104	103	105	108	103	95	100
Induction Run 2	0.8	1.0	0.9	0.9	0.8	0.9	0.9	0.9	0.7	0.8	0.7	0.7	0.97
Viability (%) Run 2	101	108	97	100	100	105	104	105	107	96	108	103
Mean Induction	1.0	1.1	1.0	1.0	1.0	1.0	1.0	1.0	0.9	1.0	0.8	0.8	1.13	n.r.
SD	0.3	0.2	0.3	0.2	0.3	0.1	0.1	0.2	0.3	0.2	0.2	0.1	0.22

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed an Imax = 1.13 (mean of two runs), and cell viability was > 70% in all runs. Based on this result, the test item is not a skin sensitiser.

Executive summary:

A KeratinoSens™ study has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item. The method was performed according to OECD 442D, under GLP. The aim of the study is to evaluate the potential of the test item to activate the Nrf2 transcription factor by quantifying the luciferase induction after exposure of these keratinocytes to the test item for 48h. 12 concentrations of the test item ranging from 0.98 to 2000 μM were prepared by serial dilution in culture medium containing 1% DMSO and added to 96-well plates of KeratinoSens cells, in two separate runs of three replicates each. Positive and negative controls were run in parallel, as well as a cytotoxicity assay (MTT reduction). All acceptance criteria were fulfilled for the positive and negative controls in each run. Under the experimental conditions described, the test item presented negative results in both runs, with a cell viability > 70% and and Imax 1.28 and 0.97 for the first and second runs, respectively. No geometric mean IC30 or IC50 were calculated since the cell viability was > 70% in both runs. Based on the test results, the test item shows no sensitisation potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22/05/2018 - 27/06/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 158: human Cell Line Activation Test (h-CLAT)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

TEST SYSTEM
- Cell line used, storage conditions and source: THP-1 (ATCC, Ref: TIB-202), stored in a cryoprotective medium in a liquid nitrogen container, from LGC Standards (Molsheim, France).
- Culture medium and conditions: cRPMI medium (RPMI 1640 with 10% FBS, 0.05 mM 2-mercaptoethanol and with penicillin and streptomycin). The cells were grown using general culture procedures and maintained in a humidified incubator set at 37ºC, 5% CO2 and were not allowed to exceed a cell density of 1 E06 cells/mL or more than 30 passages. During cell culturing, cell viability was checked using trypan blue.
- Reactivity check: Two weeks after thawing, a reactivity check was performed to qualify the cells before testing. The cell response after contact with Lactic Acid, DNCB and NiSO4. Results from reactivity check tests are compiled in CiToxLAB France internal data, with negative and positive control data obtained during each test.
- Cell culture for testing: Cells were seeded at a density between 0.1 E06 - 0.2 E06 cells/mL, and precultured in culture flasks for 48 to 72 hours, respectively. The cell density did not exceed 1 E06 cells/mL. On the day testing, cells harvested from culture flasks were re-suspended with fresh culture medium at 2 E06 cells/mL. Then, 500 μL of cells suspension were distributed into a 24-well flat bottom plate (i. e. 1 E06 cells/well).

CONTROLS
- Solvent/vehicle control: dimethylsulfoxide (DMSO, Sigma Aldrich). As DMSO was the vehicle selected at completion of the solubility assay, DMSO control formulation was included as vehicle control, and consisted in DMSO dissolved at 0.2% in cRPMI.
- Positive control: 2,4-Dinitrochlorobenzene (DNCB, Sigma-Aldrich) and Nickel Sulfate (NiSO4, Merck). As several test items were assayed concurrently, the positive controls were shared. Both positive control stock solutions were prepared within 4 hours before use, and kept at room temperature and protected from light until use.
1) DNCB was prepared at the concentration of 8 μg/mL in DMSO: on the treatment day, DNCB was mixed with DMSO at a concentration of 2 mg/mL and this solution was then 250-fold diluted in cRPMI in order to obtain a 8 μg/mL stock solution.
2) NiSO4 was prepared at the concentration of 200 μg/mL in 0.9% NaCl: on the treatment day, NiSO4 was mixed with 0.9% NaCl at a concentration of 10 mg/mL and this solution was then 50-fold diluted in cRPMI in order to obtain a 200 μg/mL stock solution.

STUDY DESIGN
- Solubility assessment: Test item solutions were prepared in saline (0.9% NaCl) and DMSO. The solubility of the test item was assessed visually for each preparation (particles, drops, cloudiness, non-miscible phases, etc) and recorded. The test item was finally found soluble in DMSO at 250 mg/mL.

- Dose Finding assay (PI Assay). 2 separate assays were conducted to assess the test item toxicity (CV75), as follows:
1) Working solutions: Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. These formulations were then diluted 250-fold (as DMSO is the selected vehicle) into cRPMI to obtain working solutions. The final concentrations used in the assays were 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125 and 250 μg/mL.
2) Assay: 500 μL of the working solutions were added to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate. The plates were incubated for 24 h ± 30 min at 37ºC and 5% CO2. At the end of the treatment phase, cells were transferred into sample tubes and
collected by centrifugation. The supernatants were discarded and the cells were re-suspended with 600 μL of FACS buffer (D-PBS with 0.1% (w/v) BSA) and the plate was put into the plate-reader of a flow cytometer. A volume of Propidium Iodine (PI) solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μ/mL per well.

- Main test (CD86/CD54 expression measurement). 2 independent validated runs were conducted as follows:
1) Working solutions: Test item formulations were prepared at 8 different concentrations by 1.2-fold dilutions in the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75. All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions. The final concentrations in the wells were (both runs): 139.5, 167.5, 200.9, 241.1, 289.4, 347.2, 416.7 and 500 μg/mL.
2) Assay: The exposure was carried out as in the Dose Finding assay. At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL of FACS buffer and blocked with 600 μL of blocking solution (0.01% globulin in FACS buffer), and incubated at 4ºC for 15 ± 1 min. After blocking, cells were split in three aliquots of 180 μL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 ± 2 min at 4ºC. Finally, cells were washed twice with 150 μL FACS buffer and re-suspended in 200 μL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 μL of PI solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of
0.625 μg/mL per well.
3) Flow cytometry analysis: After setting the aquisition channels for optimal detection (DRF), the nonspecific binding of IgG1 and CD86 and CD54 expressionwere analyzed by flow cytometry with the acquisition channel B1 in order to obtain the Mean Fluorescence Intensity (MFI); whereas the viability (PI uptake) was analyzed with the acquisition channel B3. A total of 10 000 living cells (PI negative) were acquired. When the viability was low and did not allow obtaining 10 000 living cells, a total of
30 000 events was acquired. Alternatively, cells were acquired for a maximum of 1 min after the in itiation of the acquisition. In case cell viability is less than 50%, no MFI is presented in the study report and the corresponding test item concentration are considered too high for interpretation because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.

INTERPRETATION OF RESULTS
- Description of evaluation and decision criteria used: A run conclusion is positive if at least one of the conditions below is met:
- RFI of CD86 is ≥ 150 at any concentration leading to ≥ 50% viability,
- RFI of CD54 is ≥ 200 at any concentration leading to ≥ 50% viability.

- Based on the individual run conclusions, a final prediction is made as follows:
- if the first 2 runs are both positive for CD86 and/or for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted;
- if the first 2 runs are negative for both markers, the prediction is NEGATIVE (due consideration of the highest-tested dose conditions) without the need for a third run;
- if the first 2 runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (P1) and the other is only positive for CD54 (P2), a third run is required. If this third run is negative for both markers (N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (P12), the h-CLAT prediction is considered POSITIVE.

ACCEPTANCE CRITERIA
1) Controls acceptance criteria:
- Viability of cells treated with cRPMI and DMSO controls should be ≥ 90%,
- in cRPMI and DMSO control wells, MFI ratio of both CD86 and CD54 to isotype control should be > 105%,
- in the DMSO control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI > 150% and CD54 RFI ≥ 200%),
- in the positive controls (DNCB and NiSO4), RFI values of both CD86 and CD54 should meet positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability > 50%.

2) Test item acceptance criteria:
- For a test item noted as cytotoxic in the DRF phase, and resulting in a negative outcome in the main test, cell viability at 1.2 x CV75 should be < 90% in each run,
- cell viability of at least 4 out of 8 concentrations should be > 50%.

Positive control results:

- DNCB: The RFI (CD86) was higher than 150 and the RFI (CD54) was higher than 200 in both runs, the result was considered POSITIVE.
- NiSO4: The RFI (CD86) was higher than 150 and the RFI (CD54) was higher than 200 in both runs, the result was considered POSITIVE.

Key result

Run / experiment:

other: A

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: B

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: A

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: B

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Precipitation: no abnormalities such as precipitate or cell morphology modification was observed at any tested concentrations.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS: All acceptance criteria were met in each run.
- Acceptance criteria met for negative control: Yes. Mean viability of cRPMI(1) and DMSO(1) control is > 90%, and for DMSO control(s): RFI(CD86) < 150 and RFI(CD54) < 200 with mean viability > 50%.
- Acceptance criteria met for positive control: Yes. DNCB and NiSO4 gives RFI(CD86) ≥ 150 and RFI(CD54) ≥ 200 with mean viability > 50%.

Dose Range Finding Results:

Flow cytometry measurement after Propidium Iodide staining revealed no cell viability decrease below 75% at any tested concentration in either run. Therefore, no mean CV75 value could be calculated. Based on the results from both DRF assays and from the complementary solubility test, the maximum concentration tested in the validated main test was 500 μg/mL.

Main Test Results:

Table 2. Summary of all runs and conclusion.

Test item conc. (µg/mL)	RFI for CD86		RFI for CD86		Viability (%)		Run conclusion		Conclusion
	A	B	A	B	A	B	A	B
139.5	95	93	72	89	97.0	95.0	N	N	N
167.5	87	103	20	102	96.0	96.0
200.9	91	82	92	51	96.0	95.0
241.1	73	100	60	109	97.0	95.0
289.4	82	104	96	69	97.0	94.0
347.2	71	99	60	78	96.0	94.0
416.7	71	102	64	116	97.0	94.0
500.0	75	103	84	102	97.0	95.0

N = run with negative outcome; Conc. = concentration; RFI = Relative Fluorescence Index.

Table 3. Main test individual results. Run A.

Study No.		Vehicle	Run Letter	Conc. (µg/mL)	MFI (Geo Mean)			MFI ratio		IgG corrected MFI		RFI (CD86)		RFI (CD54)		Viability (%)
												Vs. Top control		Vs. Top control
					IgG	CD86	CD54	CD86 / IgG	CD54 / IgG	CD86	CD54	vs cRPMI	vs DMSO	vs cRPMI	vs DMSO	IgG	CD86	CD54	Mean
cRPMI	(3)				1.02	2.72	1.28	267	125	1.70	0.26					96.53	97.06	96.60	97
NiSO4	(1)	0,9% NaCl		100.00	1.06	4.46	9.52			3.40	8.46	200		3254		74.24	74.64	74.87	75
0,2% DMSO	(3)				1.05	3.16	1.30	301	124	2.11	0.25	124		96		96.54	96.68	96.44	97
DNCB	(1)	0,2% DMSO		4.00	1.10	8.11	3.69			7.01	2.59		332		1036	62.10	63.84	62.77	63
46033 TIH		0,2% DMSO	A	139.54	1.06	3.06	1.24			2.00	0.18	-	95	-	72	96.66	96.59	96.68	97
				167.45	0.99	2.83	1.04			1.84	0.05	-	87	-	20	96.18	96.47	96.61	96
				200.94	0.98	2.90	1.21			1.92	0.23	-	91	-	92	95.91	96.12	95.86	96
				241.13	1.00	2.55	1.15			1.55	0.15	-	73	-	60	97.29	96.77	95.96	97
				289.35	0.91	2.63	1.15			1.72	0.24	-	82	-	96	96.54	97.04	96.48	97
				347.22	1.01	2.50	1.16			1.49	0.15	-	71	-	60	95.81	96.88	96.30	96
				416.67	0.92	2.42	1.08			1.50	0.16	-	71	-	64	96.84	96.45	96.77	97
				500.00	0.82	2.41	1.03			1.59	0.21	-	75	-	84	96.92	96.75	96.46	97

 

Table 4. Main test individual results. Run B.

Study No.		Vehicle	Run Letter	Conc. (µg/mL)	MFI (Geo Mean)			MFI ratio		IgG corrected MFI		RFI (CD86)		RFI (CD54)		Viability (%)
												Vs. Top control		Vs. Top control
					IgG	CD86	CD54	CD86 / IgG	CD54 / IgG	CD86	CD54	vs cRPMI	vs DMSO	vs cRPMI	vs DMSO	IgG	CD86	CD54	Mean
cRPMI	(2)				0.84	4.04	1.24	481	148	3.20	0.40					96.14	95.68	95.71	96
NiSO4	(1)	0,9% NaCl		100.00	0.74	6.36	13.34			5.62	12.60	176		3150		69.78	64.02	63.96	66
0,2% DMSO	(2)				0.78	4.21	1.23	540	158	3.43	0.45	107		113		94.67	95.44	95.18	95
DNCB	(1)	0,2% DMSO		4.00	0.67	7.87	3.77			7.20	3.10		210		689	75.98	77.51	75.84	76
46033 TIH		0,2% DMSO	B	139.54	0.80	3.99	1.20			3.19	0.40	-	93	-	89	95.10	95.06	94.68	95
				167.45	0.69	4.23	1.15			3.54	0.46	-	103	-	102	95.81	95.32	95.48	96
				200.94	0.75	3.57	0.98			2.82	0.23	-	82	-	51	94.52	94.99	95.07	95
				241.13	0.51	3.95	1.00			3.44	0.49	-	100	-	109	95.58	93.54	94.53	95
				289.35	0.66	4.24	0.97			3.58	0.31	-	104	-	69	94.36	93.87	93.71	94
				347.22	0.69	4.07	1.04			3.38	0.35	-	99	-	78	94.34	93.88	93.83	94
				416.67	0.72	4.22	1.24			3.50	0.52	-	102	-	116	94.54	94.39	94.23	94
				500.00	0.73	4.26	1.19			3.53	0.46	-	103	-	102	94.66	94.48	94.83	95

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found to be negative both runs up to 500 μg/mL. Therefore, the test item may be classified as not skin sensitizer.

Executive summary:

An in vitro cell line activation test (h-CLAT) has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item in accordance with the OECD Guideline 442E, under GLP. The h-CLAT method is based on changes in the quantification of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells. A solubility assay with the test item was performed, where the test item was found to be soluble at up to 250 mg/mL in DMSO, so DMSO was chosen as vehicle. Then, two Dose-Range Finding assays were performed at up to 250 μg/mL test item, where no decrease in cell viability was noted and therefore, no CV75 value could be determined. Based on the results of both DRF assays and the solubility test, the maximum concentration in the main test was 500 μg/mL. For the main test, two validated successive test runs were performed. In each run, the test item formulations (139.5, 167.5, 200.9, 241.1, 289.4, 347.2, 416.7 and 500 μg/mL) were applied to THP-1 cells and cultured for 24 hours ± 30 minutes at 37ºC, 5% CO₂ in a humidified incubator. Negative and positive controls were run in parallel. After incubation, all cells were dyed with Propinium Iodide for viability discrimination and then CD86 and CD54 expression was measured by flow cytometry analysis. The Mean Fluorescence Intensity (MFI) was obtained for each test sample, corrected by the isotype control IgG1 and compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) for CD86 and CD54 expression. All validity criteria were met. In both runs, the results of RFI(CD86) and RFI(CD54) were less than 150 and 200 respectively in all concentrations tested, both results were negative. Therefore, the test item was found to be negative in the h-CLAT.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In chemico skin sensitisation:

Weight of evidence. Study according to OECD 442C, GLP study. Based on the results of a preliminary solubility study, peptide solutions were incubated with 100 mM test item solution or 100 mM cinnamic aldehyde solution (positive control) in acetonitrile, at ratios of 1:10 for cysteine and 1:50 for lysine. Reference controls and co-elution controls were run in parallel. After 24h incubation at 25ºC, the residual peptide concentrations were evaluated by HPLC-UV (220 nm). Under test conditions, the test item co-eluted with lysine, and the % interference could not be determined, so the peptide reactivity classification was only based on the cysteine 1:10 prediction model. The percent cysteine peptide depletion for the test item was 1.06, reflecting no or minimal reactivity, and thus a negative DPRA prediction. All acceptance criteria were satisfied. Based on the test results, the test item shows no sensitisation potential.

In vitro skin sensitisation:

Weight of evidence. Study according to OECD 442D, GLP study. 12 concentrations of the test item ranging from 0.98 to 2000 μM were prepared by serial dilution in culture medium containing 1% DMSO and added to 96-well plates of KeratinoSens cells, in two separate runs of three replicates each. Positive and negative controls were run in parallel, as well as a cytotoxicity assay. All acceptance criteria were fulfilled. Under experimental conditions, the test item presented negative results in both runs, with a cell viability > 70% and Imax 1.28 and 0.97 for the first and second runs, respectively. No geometric mean IC30 or IC50 were calculated since the cell viability was > 70% in both runs. Based on the test results, the test item shows no sensitisation potential.

Weight of evidence. Study according to OECD 442E, GLP study. Based on a preliminary solubility study and two Dose-Range Finding assays, 8 test item formulations at concentrations up to 500 μg/mL in DMSO were applied to THP-1 cells and cultured for 24 hours ± 30 minutes at 37ºC, 5% CO₂. After incubation, all cells were dyed with Propinium Iodide for viability discrimination and then CD86 and CD54 expression was measured by flow cytometry analysis. The Mean Fluorescence Intensity (MFI) was obtained for each test sample, corrected by the isotype control IgG1 and compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) for CD86 and CD54 expression. Two validated test runs were performed; vehicle and positive controls were run in parallel; all validity criteria were met. In both runs, the results of RFI(CD86) and RFI(CD54) were less than 150 and 200 respectively in all concentrations tested, both results were negative. Therefore, the test item was found to be negative in the h-CLAT test.

However, according to the available evidence showing that the analogue substance piperacillin sodium salt can lead to skin sensitisation (delayed contact hypersensitivity) based on human experience (refer to Section 7.10 for more information) and also based on the evidence from chemical structure related substances (penicillins), the substance piperacillin acid might be a sensitiser. As the available information was not coherent, a confirmatory study was performed.

Weight of evidence. Study according to OECD 429, GLP study. Based on the results of preliminary tests, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each: three groups received the test item at concentrations of 50, 25, and 10% (w/v) in DMF; one negative control group received the vehicle; and one positive control group received 25 % (w/v) HCA (dissolved in DMF). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or significant systemic clinical signs were observed during the study. There were no indications of any irritancy at the site of application. The stimulation index values obtained for the test item were SI = 8.7, 8.2 and 3.0 at doses of 50, 25, and 10% (w/v), respectively, and the size of lymph nodes was in good correlation with these results. Based on these, the EC3 value of the test item was considered to be 10% (w/v). The results obtained for positive and negative controls were within historical values; all validity criteria were met. In conclusion, under the conditions of the present assay, the test item, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. According to Regulation (EC) No 1272/2008 (CLP), the test item should be classified as Category 1, sub-category 1B.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information (negative results for OECD 442C, 442D and 442E tests, but case reports indicating possible delayed contact hypersensitivity and positive result for OECD 429) the substance is proposed to be classified as Skin sensitiser Category 1B, according to CLP Regulation (EC) No. 1272/2008.