Monalazone disodium

Endpoint summary

Description of key information

DPRA: inconclusive

Keratinosens: positive

h-CLAT: positive

LLNA: weak or moderate sensitiser; EC3 value 24.33% (> 2%).

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:

15 Nov 2016 - 27 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Remarks:

The study has been conducted for US authorities originally but used here as WoE.

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Lot#: 52716BC13
Storage: RT
Purity: Mixture with 20% active ingredient (monalazone disodium)

Details on the study design:

Test & Reference Articles
Test article was received by Cyprotex lab staff and stored at room temperature. Urine was received from LeeBio Solutions and stored at 2-8°C. Positive control, 2,3-butanedione (CAS 431-03-8), was prepared at 100 mM in acetonitrile (CAS 75-05-8, Lot 150041).
Compound Name (purpose) Lot # Storage MW Purity/comments
2,3-butanedione (positive control) MKBB5095V 2 – 8°C 86.09 97% purity
Test Item (Monalazone Disodium) 52716BC13 RT NA Mixture with 20% active ingredient. See section 3c for specific formulations of test article
Fresh Human Urine (reference for urine reacted test article) W236942 (Lysine)W236942, W237946, 12A2351(Cysteine) 2 – 8°C NA Complex mixture
RT = Room Temperature

Justification of Test System
Direct Peptide Reactivity Assay: DPRA was developed by Frank Gerberick and colleagues (2004) and was further refined in 2007 (Gerberick et al., 2007), and a full OECD guideline for the assay was released in February of 2015. In this assay, the test article was incubated concurrently in two separate buffers, one with cysteine (Ac-RFAACAA-COOH) and one with lysine (Ac-RFAAKAA-COOH). Reactive chemicals bind one or both of the peptides thereby reducing their free concentration levels. The disappearance of each peptide is measured by HPLC/UV. This method does not require any biological material such as enzymes in order for this reaction to take place. It is important to note that the cysteine peptide captures soft electrophiles, while the lysine peptide captures hard electrophiles. This makes the DPRA assay a good choice to screen for reactive chemicals which are associated with allergic contact dermatitis.

Preparation of Samples
Test Article Stock: Urine was added to reactions undiluted at the same volume as the 2,3-butanedione reference chemical. In all testing conditions test article formulations were added at the same volume as the 2,3-butanedione reference chemical. The test chemical was tested in four different ways:

1. Monalazone disodium (20%)
2. A theoretical 100 mM (3.33%) active ingredient preparation prepared by mixing 4.16g 20% monalazone with ultrapure water 20.84g water
3. Monalazone disodium (20%) diluted to in use concentrations in water prepared by mixing 0.375g 20% monalazone with 24.625g ultrapure water.
4. Monalazone disodium (20%) diluted to in use concentrations in urine prepared by mixing 0.375 g 20% monalazone with 24.625 g urine.

Reference materials: 2,3-butanedione was prepared in acetonitrile (Optima LC/MS, 99.9%, Fisher, Waltham, MA, Lot No. 160783, and CAS 75-05-8) to yield a final concentration of 100 mM.

Peptides: A 0.667 mM stock solution of the lysine peptide was prepared by diluting the peptide with Lysine Reaction buffer, and a 0.667 mM stock solution of cysteine peptide was prepared by diluting the peptide in Cysteine Peptide Reaction buffer. Unprepared peptides were stored at approximately -80°C desiccated. Peptides were from LifeTein LLC (South Plainfield, NJ).
Peptide Supplier Lot Number Weight (mg) Volume (mL) Concentration
Cysteine Lifetein LT160926-LT180433 19.2 38.3 0.667mM
Lysine Lifetein LT151110-LT107617 13.2 25.5 0.667mM

Evaluation of Test Article in the Assay
DPRA: DPRA was run according to Cyprotex SOP-2078. The cysteine peptide was prepared at 0.667 mM in Cysteine Reaction buffer and the lysine peptide was prepared at 0.667 mM in Lysine Reaction buffer as outlined in OECD 442c. The reaction mix for cysteine peptide had a 1:10 test peptide to reference article ratio (0.5 mM cysteine to 5 mM reference article). The reaction mix for lysine peptide had a 1:50 peptide to reference article ratio (0.5 mM lysine to 25 mM reference article). Reactions for test and reference articles were run in triplicate. Vehicle control reactions were also made with acetonitrile or water containing no reference or test article.
Reference control reactions were prepared by mixing 2.5 mL of acetonitrile with 7.5 mL of the respective buffer. Aliquots of 1 mL were added to 9 glass vials for each peptide and placed at the beginning middle and end of the samples as shown in Annex 1. These reactions were used to ensure the consistency of peptide detection during the run.
A standard curve was prepared for both peptides by adding 400 µL of acetonitrile to 1600 µL of 0.667 mM peptide to make a 0.534 mM standard. This 0.534 mM standard was serial diluted in 20% acetonitrile/buffer to make a 6 point standard curve. A zero peptide standard was also included in the standard curve.
After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. The samples were transferred to WMed Innovation Center for peptide assessment. The Lysine reactions were analyzed via HPLC with gradient elution and UV detections at 220 nm using a Waters Alliance 2795 HPLC equipped with a 2996 Photodiode Array. Prior to sample analysis the suitability of the HPLC/UV system was verified by running the standard curve and a triplicate set of reference controls (see Annex 1). Samples were run on an Agilent Zorbax SB-C-18 column under the conditions described in section 22 of the OECD guideline. Sample analysis was initiated within 24 ±2 hours of the reaction start. The results were acquired with the MassLynx data system and quantified via the QuanLynx application. Cysteine peptide reactions were analyzed via HPLC with gradient elution and UV detections at 220 nm using a Agilent 1100 HPLC equipped with a diode array. Prior to sample analysis the suitability of the HPLC/UV system was verified by running the standard curve and a triplicate set of reference controls (see Annex 1). Samples were run on an Agilent Zorbax SB-C-18 column under the conditions described in section 22 of the OECD guideline. Sample analysis was initiated within 24 ±2 hours of the reaction start. The results were acquired with the Agilent ChemStation for LC 3D systems. Samples were injected once.
After determination of peptide remaining in the analysis, percent depletion relative to vehicle controls was calculated relative to no test article (vehicle control) samples. Peptide reactivity was reported as percent depletion and was calculated using the following formula:
% Depletion = (1-(test compound area/ vehicle control area)) x 100

Positive control results:

The mean Percent Lys Depletion for the positive control 2,3-butanedione was 22.4%.
The mean Percent Cys Depletion for the positive control cinnamic aldehyde was 81.7%.

Key result

Run / experiment:

other: 1

Parameter:

other: % Lys depletion

Remarks:

20% monalazone disodium

Value:

94.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: % Cys depletion

Remarks:

20% monalazone disodium

Value:

45.2

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Key result

Run / experiment:

other: 1

Parameter:

other: % Depletion DPRA

Value:

69.8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

Depletion of 2,3-butanedione fell into the expected in range for the assay according to the OECD guideline. The standard deviations for 2,3-butanedione and acetonitrile in the cysteine reactions were outside the expected ranges outlined in the OECD guideline and our historic observations. This is unsurprising given the behavior of the test material.

System suitability was shown by examining the r2 value for the standard curves and the average, standard deviation, and % coefficient of variation of the reference control samples. The calibration curves for both peptides were shown to have r2 values of greater than 0.99. The cysteine standard curve had an r2 value of 0.999585, while the lysine standard curve had an r2 value of 0.995273.

Reference controls containing only peptide, acetonitrile and buffer were run at the beginning, middle, and end of each peptide series to show stability. Cysteine reference controls run after the 20% monalazone samples showed a marked reduction in the amount of cysteine peptide present. Running the samples separately revealed that the reduction was due to interference from the test article.

Each test and reference article was measured in triplicate in both peptides.

Performance guidelines in OECD 442c state that Standard Deviation of the test chemical replicates should be less than 14.9% for the cysteine peptide depletion and less than 11.6% for the lysine peptide depletion. Because of the test material retention on the column, these criteria were not met for all the test and reference articles. The client test article and urine performed acceptably in the lysine reactions. Peptide depletion was noted and was proportional to the amount of test chemical added. Slight precipitation was observed in the in use urine reactions, but depletion was observed never the less.
The performance with the cysteine peptide was fairly clear that the test material is highly reactive with the cysteine peptide. However, because the analytical methodology was not ideal for the test article the study should be considered inconclusive.

Interpretation of results:

study cannot be used for classification

Conclusions:

The study should be considered inconclusive because the analytical methodology was not ideal for Monalazone disodium.

Executive summary:

The purpose of this study was to screen 20% Monalazone Disodium, for its potential to act as chemical sensitizers using the Direct Peptide Reactivity Assay (DPRA), a test used to assay reactivity of test articles with small peptides.

The test material was formulated in four different ways to analyze its properties at both bulk and in use concentrations.  A summary of the results for the test article formulations and control article is provided in table below. 

Material	%Lys Dep	%Cys Dep*	%Dep DPRA	Reactivity Class	Sensitizer
2,3-butanedione	22.4	81.7	52.0	High	Inconclusive
Urine	16.8	90.5	53.6	High	Inconclusive
20% monalazone disodium	94.4	45.2	69.8	High	Inconclusive
100 mM monalazone disodium (theoretical)	68.4	12.9	40.7	Moderate	Inconclusive
In use water 20% monalazone disodium	17.0	92.9	55.0	High	Inconclusive
In use urine 20% monalazone disodium	17.2	99.9	58.5	High	Inconclusive

*depletion calculated relative to reference controls run apart from test articles

 

An overall confounding factor for this study was the behavior of the test material in cysteine reactions during analysis. The reactions were performed three times in attempts to determine exactly what was occurring in the test system. It was observed that after running any samples containing client test material, subsequent reference reactions (containing vehicle, peptide and buffer) showed a marked decrease in peptide level compared to the expected 500 μM amount. The reference reactions were run in sets of three, and it was observed that the amount of peptide in the first replicate was very low, the next slightly higher and the final almost to the expected 500 μM level. An attempt was made to move the samples that presumably caused this issue to the end of the run; however, the issue persisted and it seems likely that any test item on the column adversely affects the downstream samples. This makes the results for the cysteine reactions highly questionable. Since there is no model using only the lysine peptide, an attempt was made to calculate the Cysteine depletion by comparing the sample data to reference controls which were run apart from the test materials. These controls showed the expected level of peptide when run prior to the test material samples, indicating they were prepared correctly but affected by the test materials when run with them. 

 

Depletion of 2,3-butanedione fell into the expected in range for the assay according to the OECD guideline. The standard deviations for 2,3-butanedione and acetonitrile in the cysteine reactions were outside the expected ranges outlined in the OECD guideline and our historic observations. This is unsurprising given the behavior of the test material. 

 

The client test article and urine performed acceptably in the lysine reactions. Peptide depletion was noted and was proportional to the amount of test chemical added. Slight precipitation was observed in the in use urine reactions, but depletion was observed never the less.  The performance with the cysteine peptide is outlined above and it was fairly clear that the test material is highly reactive with the cysteine peptide. However, because the analytical methodology was not ideal for the test chemical, the study should be considered inconclusive.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Test Item Name : Monalazone Disodium
CAS No. : Proprietary
Molecular Formula : Not available
Molecular Weight : Not available
Purity as per COA : >99% excluding water

Physical Appearance : White or Off white powder or crystals
Specific gravity : N/A
Batch No. : 72617S
Manufactured Date : 26-07-2017
Expiry date : 01-02-2019
Recommended Storage : Ambient (+18 - +36oC)
Photosensitive : Yes

Details on the study design:

Culture media preparation
Commercially available DMEM was supplemented with the following
•Fetal Bovine Serum (FBS) to a final concentration of 9.1% in medium.
•Geneticin final concentration 500 μg/mL.
The medium was stored at 2 - 8°C until use.

Treatment Medium
Commercially available DMEM was supplemented with FBS to a final concentration of 1% in the medium. The medium was stored at 2 - 8°C until use.

Solubility Test
Solubility check was done by mixing a 40 mg of the test item with 1 mL of DMSO and voxtexed, the test item was found to be partially soluble in DMSO. Solubility was then checked by mixing a 40 mg of the test item with 1 mL of sterile milli Q water and vortexed. The test item was found to be completely soluble in water.

Culture and Maintenance of KeratinoSens™ Cells
KeratinoSens™ cells were cultured and maintained in DMEM containing Glutamax supplemented with 9.1 % fetal bovine serum (FBS) and 500 μg/ml Geneticin at 37 ± 1ºC in the presence of 5% Co2. Cells were harvested for experiment when they reach 80-90% confluency. Cells were washed twice with DPBS (5 mL / flask), then 2 mL TrypLE Express Enzyme solution was added and flask were kept back into the incubator. After cells were detached (approx. 15 mins), they were resuspended in maintenance medium and splitted at a ratio of 1:3 - 1:12 in fresh medium and grown to 80-90% confluency.

Cell Seeding
•On all the experimental days, the media was replaced with fresh medium without geneticin in the morning. Cells harvested for experiment were 80 – 90 % confluent on the day of seeding and never grown to full confluency.
•The cells were washed twice with DPBS (5 mL / flask), then 2 ml TrypLE Express Enzyme solution was added and flask kept back in to the incubator. After cells have detached (approx. 15 mins), cells were re-suspended in maintenance medium without Geneticin and centrifuged at 125 g for 5 mins. Medium was aspirated and cell pellet was resuspended in maintenance medium and adjusted to a density of 80,000 cells / mL in the maintenance medium.
•A volume of 125 μl of the cell suspension (containing 10,000 cells) was added to the wells B1-B12 and H1-H11 of 96 well plates. Treatment medium not containing cells was added in H12 well of 96 well plate and kept as blank control.
•For each experiment four parallel plates were prepared: Three white 96 well plates (assay plates for Luciferase activity) and one transparent 96 well plate (cell viability assay plate for MTT assay).
•The Plates were incubated at 37 ± 1ºC in the presence of 5% CO2 for 24 ± 1h.

Test Solution Preparation
On all the days of experiment the highest concentration of test item solution 200 mM was preprared by mixing 56 mg of test item with 1 mL of Milli Q water, vortexed and filter sterilized before serially diluting.

Positive Control Solution
A volume of 5.3 µL of Cinnamaldehyde was mixed with 194.7 µL of DMSO to a stock concentration of 200 mM. A final concentration of 6.4 mM was prepared by further diluting 32 μL of the 200 mM stock solution in 968 μL of DMSO.

Preparations of 100 x master plates
The 100 × master plate was prepared from 200 mM stock solution of the test item. Test item at final concentrations of 100 mM, 50 mM, 25 mM, 12.5 mM, 6.25 mM, 3.125 mM, 1.56 mM, 0.78 mM, 0.39 mM, 0.195 mM and 0.098 mM was prepared in sterile milli Q water by Serial (2 fold) dilution of 200 mM Stock solution. The Test Compound was prepared fresh every time.

Intermediate stock Preparation (25 fold dilution in Treatment Medium)
All the master concentrations were further diluted 25-fold in treatment medium to get the intermediate concentration.

Preparation of Working stock
A 4-fold dilution of all the concentration in treatment medium was made to get the working concentrations containing 1% DMSO in final volume. The 96 well plate containing the cells were replaced with 150 µL of treatment medium and 50 µL of each concentration was added to the desired wells and mixed.

Test Item Exposure
•After the incubation period the medium was removed by aspiration and replaced with 150 μL DMEM-medium containing 1% FBS without Geneticin (Treatment medium).
•A volume of 50 μL of each concentration from the intermediate stock was added to the respective prelabelled wells (B1-B12 test item concentration ranging from 0.095 μM to 2000 μM, H1-H6 DMSO control, H7 - H11 positive concentration ranging from 4 μM to 64 μM, H1 only medium without cells) and mixed.
•All the plates were covered with a sealing tape to avoid evaporation of volatile compounds and to avoid cross-contamination between wells by volatile compounds.
•The plates were incubated at 37±1°C in presence of 5 % CO2 for 48 ± 2 h in the CO2 incubator.

Luciferase Substrate
Each bottle containing Lyophilized luciferase powder was mixed with 10 mL of luciferase assay solution

Luciferase Activity
•After the treatment exposure time, the supernatant was aspirated from the white assay plates and discarded.
•The cells wers washed by adding 100 μL of DPBS and aspirated.
•To each well, 20 μL of 1x passive lysis buffer was added (care was taken to avoid formation of foam) and the cells were incubated at Room temperature for 20 mins with gentle shaking.
•After incubation at room temperature, 50 μL of Luciferase substrate was added manually to the plates containing cell lysates and mixed. The plates with the cell lysate and the luciferase substrate were placed in the luminometer for reading.
•The luminometer was programmed to integrate the luciferase activity for 1500 ms (1.5 Seconds). The luminescence reading was saved as ‘pda’ and ‘pdf’ files for further analysis.

Viability Measurement (MTT Assay)
•For the cell viability measurement, the medium was aspirated and discarded from the transparent assay plates. A volume of 50 μL of MTT solution (1mg/mL in media) was added to all the wells (B1-B12, H1-H12).
•The plates were protected from light while adding MTT solution.
•The plates were incubated at 37 ± 1oC in presence of 5% CO2 for 4 hours.
•After incubation, the MTT medium was removed and the cells were lysed by adding 200 μL of 10 % SDS to the respective wells and incubated overnight.
•After overnight incubation, the plates were shaken for 10 mins and absorbance was read at 600 nm using photometer. The absorbance reading was saved as ‘pda’ and ‘pdf’ files for further analysis.

Positive control results:

Experiment 1: The positive control cinnamic aldehyde caused a dose related induction of the luciferase activity.The Imax was 1.79 and the EC1.5 30 μM. FAILED
Experiment 2: The positive control cinnamic aldehyde caused a dose related induction of the luciferase activity.The Imax was 3.11 and the EC1.5 37 μM. FAILED
Experiment 3: The positive control cinnamic aldehyde caused a dose related induction of the luciferase activity.The Imax was 3.89 and the EC1.5 17 μM. TRUE
Each test plate contained a five-point dose-response for the positive control cinnamaldehyde. Above indicated whether the acceptance criteria were fulfilled.
Cinnamic aldehyde induced the luciferase gene in all runs with a EC 1.5 value of 30.44, 37.14 and 16.91.
All runs were below the target of maximal 20% variability, with an % standard deviation of 12.75, 14.71 and 9.03 in the three experiments respectively.
Experiments which failed to meet all the acceptance criteria were discarded and were not considered for evaluation.

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.41

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: 1

Parameter:

other: EC1.5

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

5.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5

Value:

1.71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 250ug/ml concentration

Key result

Run / experiment:

other: 3

Parameter:

other: Imax

Value:

6.86

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 3

Parameter:

other: EC 1.5

Value:

1.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 250ug/ml concentration

Key result

Run / experiment:

mean

Parameter:

other: IC50 (ug/ml)

Value:

785

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: IC30 (ug/ml)

Value:

627

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (57 µM and 79 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.54-fold and 2.57-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.4% and 5.0% in experiment 1 and 2, respectively).

Raw Data values for Luminescence

 

Repetition	Replicate	Test item Concentrations µM
		0.098	1.95	3.9	7.8	15.6	31.25	62.5	125	250	500	1000	2000
1	1	29817	28214	25799	24187	36889	27004	32563	29655	34391	46103	15323	164
	2	28508	24200	23747	24961	26307	35188	32304	41158	36471	44128	20132	155
	3	33070	19998	57897	14032	26414	28953	23356	30309	30325	34684	15941	137
2	1	10805	3879	11419	9520	14892	12010	13726	12263	16964	65165	17240	172
	2	4707	3305	4341	5788	8342	6887	8021	6995	9940	38844	9517	97
	3	2391	2797	2661	3552	4141	7509	8054	10524	9371	19350	1847	56
3	1	4871	5612	3989	2813	4458	5942	4368	6807	3384	23899	323	45
	2	5692	5999	4601	3656	4991	6236	4709	6514	4029	22260	292	58
	3	12732	11772	6598	8696	9749	9941	5869	7594	18540	52914	262	55
Repetition	Replicate	NC	NC	NC	NC	NC	NC	CA 4 µM	CA 8 µM	CA 16 µM	CA 32 µM	CA 64 µM	Blank
1	1	31069	35861	37350	33916	29046	32352	33665	38227	48504	63182	67647	230
	2	27337	25593	24671	26313	33644	31592	30823	37004	40380	37883	52062	185
	3	27941	28489	30115	25720	26016	27012	24540	31598	35962	35471	40963	188
2	1	7116	7967	8260	6182	9707	7341	7223	7480	9075	11913	24887	137
	2	8768	6655	6511	6061	6450	6262	7022	7868	8012	10335	19743	89
	3	6470	6458	6663	6023	6286	6843	6974	8677	8651	3265	20231	51
3	1	5462	5835	5694	5855	5788	5619	3787	5038	7047	9378	20128	501
	2	5052	5585	5341	5654	5237	5619	3937	5463	7226	9502	19407	376
	3	4543	4577	4332	5265	4714	5555	4422	6112	8427	10782	18849	351

 Raw Data values for Cytotoxicity

 

Repetition	Test item Concentrations µM
	0.098	1.95	3.9	7.8	15.6	31.25	62.5	125	250	500	1000	2000
1	0.792	0.968	0.842	1.074	0.918	0.984	0.819	0.799	0.869	0.852	0.378	0.214
2	1.163	1.203	1.236	1.186	1.262	1.305	1.171	1.213	1.247	1.084	0.496	0.128
3	1.094	1.137	1.145	1.15	1.141	0.881	1.208	1.17	1.221	1.044	0.305	0.265
Repetition	NC	NC	NC	NC	NC	NC	CA 4 µM	CA 8 µM	CA 16 µM	CA 32 µM	CA 64 µM	Blank
1	0.928	1.084	1.116	1.202	1.099	1.187	1.154	1.226	1.054	1.048	1.103	0.06
2	1.725	1.268	1.473	1.161	1.162	1.144	1.093	1.171	1.185	1.089	1.119	0.062
3	1.031	0.963	0.772	1.147	1.143	1.157	0.958	1.271	1.177	1.029	0.966	0.051

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on these results it is concluded that the test item Monalazone disodium is a sensitizer in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens™ assay).

Executive summary:

The potential of the test item Monalazone Disodium to cause skin sensitisation was evaluated in ARE-Nrf2 Luciferase Test (KeratinoSens™ Assay). KeratinoSens™ assay is an in vitro cell based assay in which the KeratinoSens™ cells were exposed to the test item for 48 hours and subsequently measured for luciferase activity using a luminometer. The assay was run in 96 well plates, and test item was tested at 12 concentrations ranging form 0.98 μM to 2000 μM along with the solvent control, control blank and the reference compound cinnamic aldehyde ranging from 4 μM to 64 μM. Three white plates were tested in parallel for luciferase induction and one additional transparent plate was used for cytotoxicity assessment. The full test in triplicate analysis was independently conducted three times. The cells were exposed to the test concentrations for 48 ± 2 hours at 37 °C in a carbondioxide incubator. After the exposure time cells were lysed with passive lysis buffer, luciferase substrate was added and the luminescence activity at each concentration was integrated for 1500 ms (1.5 Seconds). The viability assay plates containing the cells were treated with treatment medium containing MTT solution for 4 hours, and then cells lysed with 10% SDS and incubated overnight. After overnight incubation, the absorbance was read at 600 nm using a photometer. The luminescence reading and the absorbance reading obtained were analyzed in an excel sheet provided by Givaduan. The test item Monalazone Disodium showed statistically significant induction above the threshold of 1.5 or 50 % at 250 and 500 µM concentration. Under the same circumstances the positive control cinnamaldehyde showed statistically significant induction over the solvent control confirming the sensitivity of the assay. Based on these results it is concluded that the test item Monalazone Disodium is a sensitizer in thein vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens™ assay).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 Oct 2018 - 31 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: . In Vitro Skin Sensitisation assay: human Cell Line Activation Test (h-CLAT). Test No. 442E, adopted: 09 October 2017.

GLP compliance:

yes

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Test Item Name : Monalazone Disodium
CAS No. : Proprietary
Molecular Formula : Not available
Molecular Weight : Not available
Purity as per COA : >99% excluding water

Physical Appearance : White or Off white powder or crystals
Specific gravity : N/A
Batch No. : 72617S
Manufactured Date : 26-07-2017
Expiry date : 01-02-2019
Recommended Storage : Ambient (+18 - +36oC)
Photosensitive : Yes

Details on the study design:

Preparation of Culture Media for Maintenance of THP-1 Cells
Media was prepared as described in the table below.
Components Volume (mL)
RPMI 1640 incomplete media 445
Fetal Bovine Serum(FBS) 50
Penicillin and Streptomycin 5
Additionally, media in culture flask was supplemented with 2- mercaptoethanol with a final concentration of 0.05 mM after every subculture.

Preparation of FACS Buffer
FACS buffer/ DPBS containing 0.1% BSA was prepared fresh on day of flow cytometry acquisition.

Preparation of Blocking Solution
Blocking solution was prepared by diluting Human BD Fc Block™ (lot 4304475) (stock concentration 500 µg/mL) on the day of use in FACS buffer.

Preparation of Propidium iodide (PI) Solution
100x concentrated PI stock solution (1.25 mg/mL) was prepared and stored in dark at 2-8oC.
On each day of the experiment 20 µL of 100x PI was diluted in 1980 µl of FACS buffer to give 12.5 µg/mL of propidium iodide working solution.

Maintenance of THP-1 Cells
THP-1 cells were cultured and maintained in RPMI-1640 media supplemented with 10% FBS and 0.05 mM 2- mercaptoethanol, containing antibiotics- Penicillin and Streptomycin at 37 ± 1ºC in the presence of 5% CO2. Cells were maintained in culture for 2 weeks before using them for the study.

Preparation of the cells for the assay: Before any assay, THP-1 cells were seeded at a density of either 0.1 × 106 cells/mL or 0.2 × 106 cells/mL and pre-cultured in culture flasks for 72 hours or for 48 hours, respectively.

Pre-test Analysis of Test System THP-1 cells: Reactivity Check
Reactivity check was carried out after 2 weeks thawing and culturing a new batch of THP-1 cells. The check was carried out using positive controls: 2, 4-dinitrochlorobenzene (DNCB), Nikel Sulphate (NiSO4) and negative control: Lactic acid (LA).
Preparation of Stock and Working Solutions for Control Chemicals
Each stock solution was kept in the dark until the working solution is prepared. Working stocks was prepared 1 hour before treatment to the cells.
• DNCB: Stock solution and working solutions was prepared as follows.
DNCB of 4.1 mg was weighed in 2 mL Eppendorf tube and 1.025 mL of DMSO was added (4 mg/mL).
Working solution (2X final conc.): Concentrations of 16, 8, 4 and 2 μg/mL were prepared by diluting the stock solution 250 times with culture medium.
• NiSO4: Stock solution and working solutions was prepared as follows.
NOSO4 of 10.4 mg was weighed in 2 mL Eppendorf tube and 1.01 mL of saline was added (10 mg/mL).
Working solution (2X final conc.): Concentrations of 200, 100, 50 and 25 μg/mL were prepared by diluting the stock solution 50 times with culture medium.
• LA: Stock solution and working solutions was prepared as follows.
To 91.9 µL of LA in 2mL Eppendorf tube, 908.1 µL of saline was added (100 mg/mL).
Working solution (2X final conc.): 2000, 1000, 500 and 250 μg/mL was prepared by diluting the stock solution 50 times with culture medium.
Treatment of Controls and CD54/CD86 Expression Measurement
Preparation of cell suspension: On the days of experiment pre-cultured cells were collected by centrifugation at 250g, 4˚C, 5 minutes and re-suspended in fresh culture medium at a density of 2 × 106 cells/mL in culture media. A volume of 80 µL of the cell suspension was added to each well of 96 well flat bottom plates (i.e. 1.6×105 cells/well).
Exposure of the chemicals and flow cytometry analysis: Equal volumes i.e 80 μL of working solutions were added to the cells, which were then cultured for 24±0.5 hours. (Final concentrations of each chemical were 8, 4,2,1 µg/mL for DNCB, 100,50,25,12.5 µg/mL for Nikel Sulphate and 1000,500,250,125 µg/mL for Lactic Acid). After incubating for 24 hours, cells were collected, stained using FITC-conjugated anti-CD86 and anti-CD54 antibodies and analyzed by flow cytometry as described in sections 7.9.5 to 7.9.10
Solvent Selection for Test Chemicals
Solubility of test item Monalazone Disodium was evaluated and confirmed visually. The test item found to be soluble in saline at 100 mg/mL concentrations. Hence, saline was selected as solvent to be used in the study.
Dose Finding for Test Item
A dose finding assay was performed to determine the test chemical concentration at which cell viability was 75% (CV75) compared to saline control/ vehicle.
Initial dose range finding experiments were carried out at concentrations ranging from 1000 µg/mL to 7.81 µg/mL with two-fold serial dilutions.CV75 value was determined to be 9.87 µg/mL and was considered as basis for dose determination and evaluation of CD54 and CD86 expression check.

Preparation of Stock and Working Solutions of Test Chemicals
Test item solution at 100 mg/mL was prepared on twice as shown in table below
S. No Date Test item (mg) Volume of Saline(µL)
1. 12-10-2018 100 1000
2. 12-10-2018 100 1000

Dilutions to prepare working solutions was carried out using the stock of 100 mg/mL as follows
Sl. No Initial concentration (µg/mL) Volume taken(µL) Volume of diluent(saline) added(µL) Intermediate stock conc.
(µg/mL)
1 100000 100 - 100000
2 100000 50 50 50000
3 50000 50 50 25000
4 25000 50 50 12500
5 12500 50 50 6250
6 6250 50 50 3125
7 3125 50 50 1562.5
8 1562.5 50 50 781.25

Futher dilution of intermediate stock of test item was carried out as in the following table.
Intermediate stock conc. (µg/mL) Volume taken (µL) Volume of culture media (µL) Working Stock (µg/mL) Volume added per well in 96 well plate (µL) Final conc. (µg/mL)
100000 5 245 2000 80 1000
50000 5 245 1000 80 500
25000 5 245 500 80 250
12500 5 245 250 80 125
6250 5 245 125 80 62.5
3125 5 245 62.5 80 31.25
1562.5 5 245 31.25 80 15.62
781.25 5 245 15.625 80 7.81

Test Item Exposure
Equal volume of working stocks i.e 80 µL was added drop wise to the cells in each well. Plates were placed in the incubator after shaking the plate gently.
Cells were exposed to 8 concentrations of test item along with negative and positive controls for 24 hours at 37ºC, 5% CO2 and humidified atmosphere in CO2 incubator along with medium and saline control.
Cell Staining with Propidium Iodide (PI)
After incubating for 24 hours (test chemical exposure), cells were transferred into 96 well round bottom plates.
Cells were washed twice with 200 µL of FACS buffer and re-suspended in 200 µL of FACS buffer.
Cells were stained by adding 10 µL of PI solution for 5-10 minutes followed by FACS analysis (final concentration of PI was 0.625 µg/mL).
Cell suspensions were transferred to 96 well round bottomed plate for FACS analysis.
Cell Viability Check by Flow Cytometry:
Flow Cytometry Acquisition was carried out using Flow cytometer BD FACSVerse™ System
The following acquisition plots were prepared:
i. 2D plot consisting of FSC (Forward Scatter) vs SSC (Side Scatter)
ii. 2D dot plot consisting of FSC vs Yellow scatter
iii. Histogram plot for Yellow scatter
A total of 10,000 living cells were acquired and the cell viability percent were shown by Flowcytometry analysis program as percent of total cell population.
Cell viability was measured by gating-out dead cells stained with PI.
Estimation of CV75 (Cell Viability 75%) Value:
The % of living cells (PI-negative cells) was used as the value for cell viability.
Using the cell viability data obtained from cytometry analysis, CV75 value was calculated using the following equation.
Log CV75 = (75 - c) × Log b – (75 - a) × Log d
a-c
Where:
a is the minimum value of cell viability over 75%
c is the maximum value of cell viability over 75%
b and d are the concentrations showing the value of cell viability a and c respectively.
The calculations were carried out using Microsoft Excel Sheet®.
Mean CV75 values of two independent runs were used for setting dose-range to measure CD86 and CD54 expression.
Measuring CD86/CD54 Expression
Day 1
Test Item Stock and Dilution Preparation
Test item stock solution was prepared on 2 days

A 100x the final concentration of the test item corresponding to 1.2 × CV75 and subsequent concentrations were prepared by diluting the 100 mg/mL as follows
100 mg stock was first diluted 1:10 times to achive 10 mg/mL concentration in Saline. Futher dilution preparation was carried out as follows

Wells Initial conc. (µg/mL) Volume taken (µL) Volume of diluent (saline) added (µL) Intermediate stock conc. (µg/mL)
1 10,000 11.6 88.4 1164
2 10,000 9.7 90.3 970
3 10,000 8.1 91.9 808
4 10,000 6.7 93.3 674
5 10,000 5.6 94.4 561
6 10,000 4.7 95.3 468
7 10,000 3.9 96.1 390
8 10,000 3.2 96.8 325
Futher dilution of intermediate stock was carried out as shown in table below.
Intermediate stock concentartion (µg/mL) Volume taken (µL) Volume of media added (µL) Working Stock (µg/mL) Volume added per well (µL) Final concentration (µg/mL)
1164 5 245 23.28 80 11.6
970 5 245 19.4 80 9.7
808 5 245 16.2 80 8.1
674 5 245 13.5 80 6.7
561 5 245 11.2 80 5.6
468 5 245 9.4 80 4.7
390 5 245 7.8 80 3.9
325 5 245 6.5 80 3.2

Cell Suspension Preparation and Seeding
THP-1 cells were prepared for the assay as described in section 7.5.
Cell Seeding: Cells were collected from culture flasks by centrifugation (approximately 250 g, 4˚C, 5 minutes) and then re-suspended in fresh culture medium at a density of 2 × 106 cells/mL. Cells were distributed into a 96 well flat-bottom plate at 80 µL/well (1.6 × 105 cells/well).
Test Item Exposure
Equal volumes (80 μL) of working solution were added to the cells, which were then cultured for 24 hours.
Final concentration of saline in each test chemical exposure wells was 1%.
In 96 well flat bottom plate formats, three replicate wells of cells were treated at each concentration separately for CD86, CD54 and Isotype control expression measurement.
Additionally, DNCB was tested as a positive control in each assay, at a final concentration of 4.0 μg/mL, 2.0 μg/mL 1.0 μg/mL and 0.5 μg/mL.
Replication
In each run, a single replicate for each concentration of the test item and control substance were analysed and a prediction was obtained from at least two independent runs.

DAY 2
Staining and Analysis
Collecting the Cells and FcR Blocking:
• The FACS buffer was prepared freshly on each day of experiment.
• For the staining and analysis step, all solutions were kept at the temperature of approximately 2 to 8°C.
• During the procedure, the cells were kept as much as possible in the dark.
Cells treated in 96 well plate were transferred to 96 well round bottom plates before blocking and staining with antibodies.
FcR blocking
Cells were blocked with 2.5 µg/L × 106 cells/100 µL/ well of blocking solution at 4ºC for 15 minutes.
Cell staining with FITC-labeled Anti CD86, CD54 Antibody
Pre-mixed antibody solution was prepared as below:
Volume of antibody Cell number Total volume of sample
FITC labelled anti-CD86 antibody 3.2 µL 1.6 X 105 50 µL
FITC labelled anti-CD54 antibody 1.6 µL 1.6 X 105 50 µL
FITC labelled mouse IgG1 1.6 µL 1.6 X 105 50 µL
A master mix of antibodies was prepared based on the number of samples to be stained. The three groups of cells were centrifuged post incubation with blocking solution and supernatant were aspirated. 50 µL each of pre-mixed antibody solution was added to respective wells. Cells were incubated at 4ºC for 30 mins in the dark.
Antibody treatment for expression analysis was carried out on 3 days (10 October 2018, 17 October 2018 and 24 October 2018. Antibodies were prepared as follows:

Total number of reactions Volume antibody stock to be added in µL FACS buffer in µL
FITC labelled anti-CD86 antibody 16 51.2 748.8
FITC labelled anti-CD54 antibody 16 25.6 774.4
FITC labelled mouse IgG1 16 25.6 774.4
Preparation of Samples for Measurement:
After staining with antibodies, the cells were washed twice with 150 µL of FACS buffer and re-suspended in a final volume of 200 µL/well FACS buffer.
10 µL of PI solution (12.5 µg/mL) was added to each well to obtain a final concentration of 0.625 µg/mL.
Flow Cytometry Acquisition:
The expression of CD86 and CD54 was measured by flow cytometry.
Preparation for acquisition:
Acquisition was carried out using BD FACSVerse™ System. Settings were adjusted for optimal detection of FITC and PI under respective voltage settings.
The following acquisition plots were prepared:
i. 2D plot consisting of FSC (Forward Scatter) vs. SSC (Side Scatter)
ii. & iii. Histogram plots of each Green and Yellow Fluorescence.
A total of 10,000 living cells were acquired and the cell viability percent showed by cytometry analysis program as percent of total cell population.

Acquisition
• Cell Debris and Dead cells were gated-out by staining with PI.
• Total of 10,000 living cells were analyzed.
• Mean fluorescence intensity (MFI) of viable cells and viability for each sample was used for analysis.
• When the cell viability was less than 50%, the relative fluorescence intensity (RFI) was not used because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.

Flow cytometry analysis
Mean Fluorescence Intensity (MFI) or the Geometric mean was calculated using FlowJo Software.
The Relative Fluorescence Intensity (RFI) was used as an indicator of CD86 and CD54 expression, and was calculated as follows for each concentration:
RFI= MFI of chemical treated cells – MFI of chemical treated isotype cells ×100
MFI of Solvent treated control cells – MFI of solvent treated isotype cells
• For each concentration of every chemical, the cell viability was recorded from the isotype control cells (stained with FITC labelled-mouse IgG1).
• When the cell viability was less than 50%, the relative fluorescence intensity (RFI) was not used because of the diffuse labelling cytoplasmic structures that are generated following cell membrane destruction.

Calculation of EC150 and EC200
As the RFI value at the lowest dose is above the positive criteria in both CD86 and CD54 expression, the EC150 (CD86) or EC200 (CD54) values by log-linear extrapolation was not calculated. Hence, based upon on the results obtained, it can be predicted that the EC150 and EC200 values of test item is below 3.2 µg/mL.

Acceptance criteria:
Following acceptance criteria was applied to the test runs in evaluation of the test item MONALAZONE DISODIUM.
• Cell viability of more than 90% for medium and DMSO controls
• RFI values indicating CD86 ≥ 150 and CD54 ≥ 200 for positive control (DNCB) and cell viability of more than 50%
• In solvent/vehicle control, RFI values compared to the medium control of both CD86 and CD54 not exceeding the positive criteria (CD86 ≥ 150 and CD54 ≥ 200).
• For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control being > 105%.
• For the test chemical, the cell viability to be more than 50% in at least four tested concentrations in each run.
Test results which did not meet the above acceptance criteria were not considered.

Positive control results:

CD86 CD54
Sample ID RFI % viability RFI % viability

Positive control
DNCB - 2µg/mL 172 83.6 426 93.8

Positive Control
NiSO4 -50µg/mL 758 89.8 778 92.1

Key result

Run / experiment:

other: ug/ml

Parameter:

other: CV75

Value:

9.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: RFI/CD86

Remarks:

Monalazone Disodium 3.2 µg/mL

Value:

176

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: RFI/CD86

Remarks:

Monalazone Disodium 3.2 µg/mL

Value:

190

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: RFI/CD54

Remarks:

Monalazone Disodium 3.2 µg/mL

Value:

403

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: RFI/CD54

Remarks:

Monalazone Disodium 3.2 µg/mL

Value:

564

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent/vehicle control: YES, RFI values compared to the medium control of both CD86 and CD54 not exceeding the positive criteria (CD86 ≥ 150 and CD54 ≥ 200).
- Acceptance criteria met for positive control: YES, RFI values indicating CD86 ≥ 150 and CD54 ≥ 200 for positive control (DNCB) and cell viability of more than 50%

Two experimental repeats of CD86 and CD54 expression check were carried out. Results of the two experiments are presented in tables below

Measurement of CD86 and CD54 Expression: Experiment-1  

parameter	MFI		RFI	Cell Viability (%)
	CD86	Isotype Control
DMSO Control	89	43.7	100.00	92.1
Saline Control	82.8	42.4	100.00	92.9
Monalazone Disodium   3.2 µg/mL	152	80.8	176.24	86.7
Monalazone Disodium   3.9 µg/mL	152	82.5	172.03	86.8
Monalazone Disodium     4.7 µg/mL	157	84.5	179.46	86.2
Monalazone Disodium      5.6 µg/mL	163	85.9	190.84	85.4
Monalazone Disodium   6.7 µg/mL	165	83	202.97	85.4
Monalazone Disodium     8.1 µg/mL	157	112	111.39	54.9
Monalazone Disodium   9.7 µg/mL	160	87.6	179.21	56.8
Monalazone Disodium     11.6 µg/mL	154	83.4	174.75	60.4
DNCB  2 µg/mL	198	85.1	249.23	53.2
Well parameter	MFI		RFI	Cell Viability (%)
	CD54	Isotype Control
DMSO Control	82	43.7	100.00	90.2
Saline Control	80.9	42.4	100.00	90.4
Monalazone Disodium   3.2 µg/mL	236	80.8	403.12	86.1
Monalazone Disodium   3.9 µg/mL	228	82.5	377.92	86
Monalazone Disodium     4.7 µg/mL	234	84.5	388.31	84.7
Monalazone Disodium      5.6 µg/mL	234	85.9	384.68	84.8
Monalazone Disodium   6.7 µg/mL	232	83	387.01	85
Monalazone Disodium     8.1 µg/mL	234	112	316.88	51.2
Monalazone Disodium   9.7 µg/mL	240	87.6	395.84	52.4
Monalazone Disodium     11.6 µg/mL	246	83.4	422.34	54.5
DNCB  2µg/mL	184	85.1	258.22	49.6

Measurement of CD86 and CD54 Expression: Experiment-2

 

Well parameter	MFI		RFI	Cell Viability (%)
	CD86	Isotype Control
DMSO Control	131	78	100.00	94.2
Saline Control	81.2	54.3	100.00	95
Monalazone Disodium   3.2 µg/mL	130	78.8	190.33	90.2
Monalazone Disodium   3.9 µg/mL	130	78.6	191.08	86.6
Monalazone Disodium     4.7 µg/mL	131	79.8	190.33	83.5
Monalazone Disodium      5.6 µg/mL	129	79.1	185.50	89.1
Monalazone Disodium   6.7 µg/mL	137	80.6	209.67	83.1
Monalazone Disodium     8.1 µg/mL	140	79.5	224.91	80
Monalazone Disodium   9.7 µg/mL	136	79.7	209.29	74.6
Monalazone Disodium     11.6 µg/mL	119	80.3	143.87	67.4
DNCB  2µg/mL	186	93.3	174.91	69.6
Well parameter	MFI		RFI	Cell Viability (%)
	CD54	Isotype Control
DMSO Control	112	78	100.00	94.2
Saline Control	80	54.3	100.00	93
Monalazone Disodium   3.2 µg/mL	224	78.8	564.98	89.7
Monalazone Disodium   3.9 µg/mL	225	78.6	569.65	85.6
Monalazone Disodium     4.7 µg/mL	226	79.8	568.87	84.1
Monalazone Disodium      5.6 µg/mL	228	79.1	579.38	88.6
Monalazone Disodium   6.7 µg/mL	234	80.6	596.89	81
Monalazone Disodium     8.1 µg/mL	234	79.5	601.17	79.8
Monalazone Disodium   9.7 µg/mL	233	79.7	596.50	78.7
Monalazone Disodium     11.6 µg/mL	229	80.3	578.60	68.4
DNCB  2µg/mL	193	93.3	293.24	73.1

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Monalazone Disodium is a skin sensitizer by hCLAT method.

Executive summary:

Human Cell Line Activation Test (hCLAT) was carried out according to test guidelines OECD 442E to evaluate skin sensitization potential of the test item Monalazone Disodium. Test system (THP-1) suitability was first evaluated by reactivity check using positive controls 2,4-dinitrochlorobenzene (DNCB) & Nickel Sulphate (NiSO4) and negative control Lactic acid (LA). DNCB and NiSO4produced a positive response (increase in expression of CD86 and CD54 cell surface markers) and hence this experiment demostrated the suitability of the test system to evaluate the test item. The test item was evaluated in a 3-step procedure. First by determining the solubility, followed by determination of CV75 value (Test Item concentration at which viability of THP-1 cells is reduced to 75%) to dermine the dose range to be tested, followed by evaluation for CD86 and CD54 expression.

The test item Monalazone Disodium was found to be soluble in saline at concentration of 100 mg/mL. Average CV75 value was determined to be 9.7 µg/mL. Based on the CV75 eight concentrations /doses of 11.6, 9.7, 8.1, 6.7, 5.6, 4.7, 3.9 and 3.2 µg/mL was tested for CD86 and CD54 expression. Expression levels of CD86 and CD54 were determined by Mean Fluorescense Intensities (MFI) values. Relative Fluorescence Intensities (RFI) values were calculated based on MFI values of test samples relative to vehicle control and isotype controls. Based in the calculated RFI values Test Item Monalazone Disodium showed increase in both CD86 and CD54 expression even at lowest tested dose 3.2 µg/mL in repeat experiments. At the concentration, 3.2µg/mL where the RFI values of CD86 and CD54 was found to be greater than 150 and 200, respectively, and the viability of THP-1 was greater than 50%. Based on the results obtained for Monalazone Disodium, it can be concluded that the Monalazone Disodium is a skin sensitizer by hCLAT method.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 January 2019 - 13 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Test Item Name : Monalazone Disodium

CAS No. : Proprietary
Molecular Formula : C7H4ClNNa2O4S
Molecular Weight : 279.602 g/mol
Purity as per CoA : >99% excluding water

Physical Appearance : White or Off white powder or crystals
Aqueous pH : 8-10
Lot No. : 72617S
Manufactured Date : 26-07-2017
Expiry date : 28/02/2019

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation:
18.9 to 21.8 grams
- Housing:
Animals were housed individually (to avoid licking of test item by cage mates) in solid floor standard polysulfone cages (Size: Approximately L 360 x B 205 x H 140 mm), with stainless steel top grill
- Diet (e.g. ad libitum):
ad libitum Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet-Pellet (Certified) manufactured by Envigo, P.O. Box 44220, Madison, WI 53744-4220
- Water (e.g. ad libitum):
deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier (manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India).
- Acclimation period:
6 days before start of the treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C
- Humidity (%): 65-67 %
- Air changes (per hr): 13.3 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: 1% Pluronic® L92

Concentration:

The substance was tested at three concentrations (15 %, 30 % and 60 %)

No. of animals per dose:

6 animals / dose

Details on study design:

Group Allocation and Number of Animals
The selected female mice were assigned to groups as shown below:

Group Dose concentration Vehicle Volume Applied to each ear Sex No. of animals Animal Nos.

G1
Vehicle Control 0 1% L92 25 µL F 6 Mb8841 to Mb8846

G2
Positive Control
α-hexylcinnamaldehyde (HCA) 25 % v/v 1% L92 25 µL F 6 Mb8847 to Mb8852

G3 15 % w/v 1% L92 25 µL F 6 Mb8853 to Mb8858

G4 30 % w/v 1% L92 25 µL F 6 Mb8859 to Mb8864

G5 60 % w/v 1% L92 25 µL F 6 Mb8865 to Mb8870

1% L92: 1% Pluronic® L92

Performance of Test

Dose preparation and analysis
Concentrations tested for the irritancy screen were selected based upon miscibility / solubility in an appropriate LLNA vehicle. The toxicity data regarding the irritation potential was also taken into consideration.
The required quantity of the test item was mixed with 1% L92 to obtain a stock formulation with concentration of 60% w/v. The required volume of stock formulation was mixed with 1% L92 to get dose formulations concentrations of 15 and 30% w/v. The dose formulations were prepared daily just prior to dosing. Preparation of the dosing materials was documented in the study file. The concentrations of the dose formulations were not verified analytically.

Irritation Screen
A concentration of 60% w/v was the maximum soluble concentration in 1% Pluronic® L92 (1% L92). Hence the highest possible dose concentration of 60% w/v in 1% L92 was selected as highest dose concentration for testing.
Prior to the LLNA main study, the vehicle 1% L92 and concentrations of 5, 10, 30, 45 and 60% w/v in 1% L92 were evaluated for irritation potential as measured by erythema of the ears.
Both ears of six female mice (one mouse/concentration) were topically treated once daily for three consecutive days with one of the concentrations of the test item.
The test item was applied using an adjustable micropipette with disposable tips. All mice received 25 µL of one concentration of the test item, spread over the dorsal surface of each ear in a manner to prevent test item loss (50 µL total / mouse). Similarly, the vehicle was applied to the ears of one animal. Both ears were observed for skin reaction prior to application of the test item (on day 1, 2 and 3), and on day 6. All mice were weighed on Days 1 and 6. Ear thickness was measured using Digimatic micrometer (Mitutoyo, Japan) prior to dosing on Days 1 and 3 and on day 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determination as detailed in paragraph 11.6, after animals were euthanized. Erythema scores, ear thickness and body weight data following the test item applications were compared to the response of the animals treated with vehicle alone. Irritation to mouse ears is only relevant in the context of the LLNA and should not be interpreted as an indicator for an irritation potential in humans.
From the results of this screening study, the main study dose concentrations of 15, 30 and 60% w/v test item in 1% L92 were selected, because none of the tested concentrations elicited any irritation reaction, no increased ear thickness and ear punch weights.
Note: Ear thickness and ear punch weight were determined for irritation screen only.

Main Study, Dermal Sensitization
The application of the test item (25 µL/ear) was made on the dorsum of both ears as described above. Six female mice/group received the vehicle (1% L92, or the positive control substance (25% v/v α-hexylcinnamaldehyde), or 15, 30 and 60% w/v test item in 1% L92, once daily for three consecutive days. Both ears were observed for skin reaction prior to application of the test item (on day 1, 2 and 3), and on day 6. All mice were weighed on days 1 and 6.

Preparation of 3H-Methyl Thymidine (3H -TdR)
A solution of 3H-TdR (80 µCi/mL)[specific activity 6.7 Ci/mmol; Perkin Elmer, USA] in sterile phosphate buffer saline (PBS) was prepared freshly. The prepared working solution of 3H-TdR was analyzed for radioactivity.

Injection of 3H-Methyl Thymidine (3H -TdR)
On day 6, a volume of 250 µL (20 µCi) of 3H-TdR in PBS was administered to each mouse via the lateral tail vein using a 1.0 mL disposable syringe fitted with 26 G x ½ inch needle.

Collection of auricular lymph nodes
Approximately five hours post injection of 3H-TdR, animals were euthanized using isoflurane anaesthesia. The auricular lymph nodes (bilateral) were excised and placed in PBS, processed (as described in 11.7) and the radioactivity was counted.

Ear punch weight
The animals were euthanized using isoflurane anesthesia and both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 6 mm). For each animal both punches were immediately weighed using an analytical balance.

Processing of auricular lymph nodes
A single cell suspension of the lymph node cells (LNC) from each mouse was prepared by gentle mechanical disaggregation using a tissue homogeniser (Stomacher 80 MicroBiomaster, Seward Ltd, United Kingdom) for 30 seconds at medium speed using PBS (approx. 10 mL).
After all the nodes had been processed, the tubes were centrifuged at 200 x g for 10 minutes at 4°C. The supernatant was poured into a container for radiolabel waste collection. 10 mL of PBS was added to each tube and inverted to resuspend the pellet. The tubes were centrifuged as described above and the supernatant was poured off. After the second wash, the cell pellet was suspended in 3.0 mL of 5% trichloroacetic acid (TCA) and stored overnight at 2-8°C for approximately 19 hours. Clumping of LNC was avoided by ensuring that the pellet was completely resuspended in a small volume of PBS before making up to the final volume. The suspended precipitates were centrifuged at 200 x g for 10 minutes at 4°C and the supernatant was poured off into container for radiolabel waste collection. The pellet from each mouse was reconstituted in 1 mL of 5% TCA and subsequently transferred to a scintillation vial containing 10 mL of a scintillation cocktail (Insta-Gel Plus, PerkinElmer, USA). Two additional 2 mL aliquots of water were used to rinse the tubes and the rinses were added to the scintillation vial containing 1 mL of the pellet in TCA and cocktail. The samples were mixed using a snapping wrist action.
The radioactivity in each precipitate was measured for 5 minutes using a ß-scintillation counter (Tricarb 2900-TR, Packard Instruments, USA) as disintegrations per minute (dpm) per mouse.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A mean dpm value ± SD (standard deviation) was calculated for each group and the stimulation index (SI) was calculated using the absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle-treated mice as the denominator.

1. The % increase in ear thickness was calculated for each ear using the following equation:
% Ear swelling = (B – A) / A x 100
Where, A = ear thickness measurement on Day 1 (µm); B = ear thickness measurement on Day 3 or 6 (µm)

2. The SI was calculated for each mouse using the following equation:
SI = Disintegrations per minute (dpm) of individual mouse / Average dpm of the vehicle control mice

3. EC3 calculation:
EC 3 = XL + [(3-YL)/ (Yh-YL)](Xh-XL)
Where, YL = SI value below 3
XL = chemical concentration that elicits YL
Yh = SI value above 3
Xh = chemical concentration that elicits Yh

EC3 : Estimated concentration of the chemical necessary to give a 3-fold increase in the lymph node cell proliferative activity compared to vehicle- treated group (SI ≥ 3).

Means and SD were generated for body weight data (absolute and gain) and LLNA response (dpm and SI values). The body weight and dpm data were analysed by one-way analysis of variance. When the differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett’s t-test (p<0.05).

Statistically significant differences (p<0.05), indicated by the aforementioned tests, are designated by the superscripts throughout the report as stated below:
+/- : Significantly higher (+) / lower (-) than the vehicle control group

Positive control results:

The sensitivity of this LLNA test was demonstrated via the response from the positive control (25% HCA in 1% L92), which elicited a stimulation index (SI) of 8.08, in comparison with the vehicle-treated mice.

Key result

Parameter:

SI

Value:

2.44

Test group / Remarks:

15% test item

Key result

Parameter:

SI

Value:

3.34

Test group / Remarks:

30% test item

Key result

Parameter:

SI

Value:

4.39

Test group / Remarks:

60% test item

Key result

Parameter:

EC3

Value:

> 24.33

Cellular proliferation data / Observations:

Summary of Disintegrations Per Minute (DPM) for 3H-Methyl Thymidine Incorporation in Auricular Lymph Nodes and Stimulation Index (SI)

Group and
Dose concentration No. of mice DPM / Mouse SI

G1
Vehicle: 1% L92 6 Mean 948.17 1.00
SD 152.93 0.16


G2
25% v/v HCA 6 Mean 7661.33 + 8.08
SD 2032.09 2.14


G3
15% w/v test item 6 Mean 2318.67 + 2.44
SD 296.05 0.31


G4
30% w/v test item 6 Mean 3162.17 + 3.34
SD 870.80 0.92


G5
60% w/v test item 6 Mean 4161.33 + 4.39
SD 1162.40 1.23

+: Significantly higher than the vehicle control group

1% L92 : 1% Pluronic® L92
HCA : α – Hexylcinnamaldehyde

TABLE 1.       Summary of Body Weight and Body Weight Changes   Group and Dose concentration   No. of mice   Body weight (g) Day 1 (Initial)     Day 6 Weight change (day 6 – Initial) Clinical signs   G1 Vehicle: 1% L92   6           Mean 20.25 20.73 0.48 NAD SD 0.99 1.03 0.08             G2 25% v/v HCA   6           Mean 20.23 20.70 0.47 NAD SD 0.89 0.87 0.10             G3 15% w/v test item   6           Mean 20.10 20.63 0.53 NAD SD 0.81 0.90 0.15             G4 30% w/v test item   6           Mean 20.12 20.58 0.47 NAD SD 0.83 0.83 0.05             G5 60% w/v test item   6           Mean 20.08 20.57 0.48 NAD SD 0.77 0.80 0.04           1% L92 : 1% Pluronic^®L92 NAD    : No Abnormality Detected HCA           : α – Hexylcinnamaldehyde       TABLE 2.       Summaryof Local Reaction Scores at the Site of Application                                                                                   Group and Dose concentration   No. of Mice   Erythema Score of both ears (Mean ± SD) Pre-treatment   Day 2 Day 3 Day 6 G1 Vehicle: 1% L92 6 Mean SD 0 0 0 0 0 0 0 0 G2 25% v/v HCA 6 Mean SD 0 0 0.83 0.41 1.00 0.00 1.00 0.00 G3 15% w/v test item 6 Mean SD 0 0 0 0 0 0 0 0 G4 30% w/v test item 6 Mean SD 0 0 0 0 0 0 0 0 G5 60% w/v test item 6 Mean SD 0 0 0 0 0 0 0 0 1% L92 : 1% Pluronic^®L92 HCA : α – Hexylcinnamaldehyde

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The skin sensitising potential of Monalazone Disodium was assessed using murine local lymph node assay (LNNA). As EC3 value was The substance is weak (Ecetoc) or moderate (Basketter) sensitiser in the LLNA study under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate whether the substance induces skin sensitization in mice after three epidermal exposures of the animals

In the Irritation screening test once daily topical applications of vehicle- 1% Pluronic^®L92 (1% L92), 5, 10, 30, 45 and 60% w/vtest itemin 1% L92 were performed to one animal at each dose level for 3 days. There were no clinical signs, no erythema at the site of application, no significant increase in the ear thickness and ear punch weights and no effect on body weight (Table 1). Results from this screening study were used to determine the dosing concentrations of Monalazone Disodium  for the main LLNA study.

The analyzed radioactivity of ³H-TdR working solution was 76.82 µCi/mL against the nominal concentration of 80 µCi/mL. There were no clinical signs,no erythema at the site of applicationand no significant effect on body weight gains. The sensitivity of this LLNA test was demonstrated via the response from the positive control (25% HCA in 1% L92), which elicited a stimulation index (SI) of 8.08, in comparison with the vehicle-treated mice. The Mean SI values for 15, 30 and 60% (w/v) test item in 1% L92 were 2.44, 3.34 and 4.39, respectively. The EC₃value was 24.33%.

The test item Monalazone Disodium is categorised as ‘Weak sensitizer’, as per the ECETOC categorisation for relative skin sensitization potency or moderate sensitiser as described by Basketter et al. (2005).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

WoE approach was used to assess sensitisation potential of the substance.

DPRA (OECD 442C)

The purpose of this study was to screen (20% Monalazone Disodium), for its potential to act as chemical sensitizers using the Direct Peptide Reactivity Assay (DPRA), a test used to assay reactivity of test articles with small peptides.

The material was formulated in four different ways to analyze its properties at both bulk and in use concentrations.  A summary of the results for the test article formulations and control article is provided in table below. 

.

Material	%Lys Dep	%Cys Dep*	%Dep DPRA	Reactivity Class	Sensitizer
2,3-butanedione	22.4	81.7	52.0	High	Inconclusive
Urine	16.8	90.5	53.6	High	Inconclusive
20% monalazone	94.4	45.2	69.8	High	Inconclusive
100 mM monalazone (theoretical)	68.4	12.9	40.7	Moderate	Inconclusive
In use water	17.0	92.9	55.0	High	Inconclusive
In use urine	17.2	99.9	58.5	High	Inconclusive

*depletion calculated relative to reference controls run apart from test articles

 

An overall confounding factor for this study was the behavior of the test material in cysteine reactions during analysis. The reactions were performed three times in attempts to determine exactly what was occurring in the test system. It was observed that after running any samples containing client test material, subsequent reference reactions (containing vehicle, peptide and buffer) showed a marked decrease in peptide level compared to the expected 500 μM amount. The reference reactions were run in sets of three, and it was observed that the amount of peptide in the first replicate was very low, the next slightly higher and the final almost to the expected 500 μM level. An attempt was made to move the samples that presumably caused this issue to the end of the run; however, the issue persisted and it seems likely that any test material on the column adversely affects the downstream samples. This makes the results for the cysteine reactions highly questionable. Since there is no model using only the lysine peptide, an attempt was made to calculate the Cysteine depletion by comparing the sample data to reference controls which were run apart from the test materials. These controls showed the expected level of peptide when run prior to the test material samples, indicating they were prepared correctly but affected by the test materials when run with them. 

 

Depletion of 2,3-butanedione fell into the expected in range for the assay according to the OECD guideline. The standard deviations for 2,3-butanedione and acetonitrile in the cysteine reactions were outside the expected ranges outlined in the OECD guideline and our historic observations. This is unsurprising given the behavior of the test material. 

 

The client test article and urine performed acceptably in the lysine reactions. Peptide depletion was noted and was proportional to the amount of test chemical added. Slight precipitation was observed in the in use urine reactions, but depletion was observed never the less.  The performance with the cysteine peptide is outlined above and it was fairly clear that the test material is highly reactive with the cysteine peptide. However, because the analytical methodology was not ideal for the test chemical, the study should be considered inconclusive.

Keratinosens (OECD 442D)

The potential of the test item Monalazone Disodium to cause skin sensitisation was evaluated in ARE-Nrf2 Luciferase Test (KeratinoSens™ Assay). KeratinoSens™ assay is anin vitrocell based assay in which the KeratinoSens™ cells were exposed to the test item for 48 hours and subsequently measured for luciferase activity using a luminometer. The assay was run in 96 well plates, and test item was tested at 12 concentrations ranging form 0.98 μM to 2000 μM along with the solvent control, control blank and the reference compound cinnamic aldehyde ranging from 4 μM to 64 μM. Three white plates were tested in parallel for luciferase induction and one additional transparent plate was used for cytotoxicity assessment. The full test in triplicate analysis was independently conducted three times. The cells were exposed to the test concentrations for 48 ± 2 hours at 37 °C in a carbondioxide incubator. After the exposure time cells were lysed with passive lysis buffer, luciferase substrate was added and the luminescence activity at each concentration was integrated for 1500 ms (1.5 Seconds). The viability assay plates containing the cells were treated with treatment medium containing MTT solution for 4 hours, and then cells lysed with 10% SDS and incubated overnight. After overnight incubation, the absorbance was read at 600 nm using a photometer. The luminescence reading and the absorbance reading obtained were analyzed in an excel sheet provided by Givaduan. The test item Monalazone Disodium showed statistically significant induction above the threshold of 1.5 or 50 % at 250 and 500 µM concentration. Under the same circumstances the positive control cinnamaldehyde showed statistically significant induction over the solvent control confirming the sensitivity of the assay. Based on these results it is concluded that the test item Monalazone Disodium is a sensitizer in thein vitroskin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens™ assay).

h-CLAT (OECD442E)

Human Cell Line Activation Test (hCLAT) was carried out according to test guidelines OECD 442E to evaluate skin sensitization potential of the test item Monalazone Disodium. Test system (THP-1) suitability was first evaluated by reactivity check using positive controls 2,4-dinitrochlorobenzene (DNCB) & Nickel Sulphate (NiSO4) and negative control Lactic acid (LA). DNCB and NiSO4produced a positive response (increase in expression of CD86 and CD54 cell surface markers) and hence this experiment demostrated the suitability of the test system to evaluate the test item. The test item was evaluated in a 3-step procedure. First by determining the solubility, followed by determination of CV75 value (Test Item concentration at which viability of THP-1 cells is reduced to 75%) to dermine the dose range to be tested, followed by evaluation for CD86 and CD54 expression.

The test item Monalazone Disodium was found to be soluble in saline at concentration of 100 mg/mL. Average CV75 value was determined to be 9.7 µg/mL. Based on the CV75 eight concentrations /doses of 11.6, 9.7, 8.1, 6.7, 5.6, 4.7, 3.9 and 3.2 µg/mL was tested for CD86 and CD54 expression. Expression levels of CD86 and CD54 were determined by Mean Fluorescense Intensities (MFI) values. Relative Fluorescence Intensities (RFI) values were calculated based on MFI values of test samples relative to vehicle control and isotype controls. Based in the calculated RFI values Test Item Monalazone Disodium showed increase in both CD86 and CD54 expression even at lowest tested dose 3.2 µg/mL in repeat experiments. At the concentration, 3.2µg/mL where the RFI values of CD86 and CD54 was found to be greater than 150 and 200, viability of THP-1 was greater than 50%. Based on the results obtained for Monalazone Disodium, it can be concluded that the Monalazone Disodium is a skin sensitizer by hCLAT method.

LLNA (OECD 492)

The purpose of this Local Lymph Node Assay (LLNA) was to assess the potential of the test item to cause contact sensitization via measurement of lymph node proliferation following topical application of the test item to the dermal surfaces of the mouse ear. The irritation potential was evaluated prior to conducting the LLNA to identify potential confounding test item concentrations that might produce excessive skin irritation to mice. Indications of ear irritation are only relevant in the context of LLNA and should not be interpreted as an indication for an irritation potential in humans. The sensitizing potential was subsequently determined by the magnitude of the lymphocyte proliferative response in the auricular lymph nodes draining the ears. A test item that elicits a stimulation index [SI of>3 (i.e., 3-fold greater proliferation than vehicle control treated animals)] should be considered positive for a dermal sensitization potential.

There were no clinical signs, no erythema at the site of application and no significant effect on body weight gains. The sensitivity of this LLNA test was demonstrated via the response from the positive control (25% HCA in 1% L92), which elicited a stimulation index (SI) of 8.08, in comparison with the vehicle-treated mice. The Mean SI values for 15, 30 and 60%w/v test item in 1% L92 were 2.44, 3.34 and 4.39, respectively. The EC₃value was 24.33%. The test item Monalazone Disodium is categorised as ‘Weak sensitizer’, as per the ECETOC categorisation for relative skin sensitization potency or "moderate sensitiser" per Basketter et al. (2005).

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on WoE approach the substance need to be classified for Skin Sens 1B according to the CLP Regulation No. 1272/2008.