Calcium disulphamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Calcium disulphamate

EC Number:

237-399-8

EC Name:

Calcium disulphamate

Cas Number:

13770-92-8

Molecular formula:

Ca.2H3NO3S

IUPAC Name:

calcium disulfamate

Specific details on test material used for the study:

Name of test substance: Calcium disulphamate
Test-substance No.: 16/0399-1
Batch identification: GM0056-WRS-0220
CAS No.: 13770-92-8
Content: 90.9 g/100 g (see report no.: 16L00478) 90.9% was used for calculation of 100 mM concentration (DPRA) and maximum concentration used for LuSens and h-CLAT)
Identity: confirmed (see report no.: 16L00478)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
Physical state / color: solid, white
Storage conditions: ambient
Molecular weight: 232.25 g/mol
Log KOW: -2.6 (calculated)
Proposed reaction mechanism for protein binding by OECD toolbox (non-GLP system): The OECD toolbox did not indicate an alert for protein binding for either the substance or its predicted metabolites (auto-oxidation, hydrolysis, and skin metabolism).

In vitro test system

Details on the study design:

Dendritic cell activation has been identified as one of the key events of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10). The human cell line activation test (h-CLAT) is a dendritic cell activation test to predict skin sensitizing potential (Ashikaga et al. 2010, Sakaguchi et al. 2010). The test is performed using the human monocytic leukemia cell line THP-1 as surrogate for dendritic cells.
As readout, the change in the expression of the cell membrane markers CD 54 and CD 86 measured by flow cytometry after 24 hours of test substance exposure is determined.
Cell line: THP-1 cells
The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB-202).

Results and discussion

In vitro / in chemico

Results

Results 1

Parameter:

other: % relative fluorescence intensity CD86

Value:

150

Vehicle controls validity:

valid

Results 2

Parameter:

other: % relative fluorescence intensity for CD54

Value:

200

Vehicle controls validity:

valid

Any other information on results incl. tables

For detailed results see attached document.

Acceptance criteria and historic control data of the h-CLAT

The acceptance criteria mentioned above were met in all experiments. The positive and negative and vehicle control data is comparable to historic data as can be seen in the following table:

Table: Historic control data of h-CLAT. Data shown of test period Jan 2016 until Jun 2017 (not including present study).

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viabilitymean[%]		rel. viability mean [%]
Min	31	43		95		99
Max	134	184		99		101
Mean	71	112		98		100
SD	11	18		1		0
n (experiments)			143

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viabilitymean[%]		rel. viability mean [%]
Min	151	211		71		73
Max	528	1893		92		98
Mean	286	591		84		86
SD	64	270		4		4
n (experiments)			143

VehicleControl (culturemedium)	CD86 RFI [%]	CD54 RFI [%]		viabilitymean[%]
Min	56	68		95
Max	144	132		99
Mean	100	100		98
SD	20	13		1
n (experiments)			143

Vehicle Control (DMSO)	CD86 RFI [%]	CD54 RFI [%]		viabilitymean[%]		rel.viabilitymean[%]
Min	39	48		90		92
Max	150	189		99		101
Mean	107	107		98		100
SD	21	18		1		1
n (experiments)			143

Table: Reactivity check, performed with each new-thawed cells prior to use for a study, using NiSO4, LA, DNCB and the vehicle control (data shown of test period Mar 2015 until Jul 2017).

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	rel.viabilitymean[%]
Min	62	69	99
Max	90	169	101
Mean	72	111	100
SD	8	25	0
n (experiments)		18

Nickel(II)sulfatehexahydrate (NiSo4100µg/mL)	CD86 RFI [%]	CD54 RFI [%]	viability mean [%]
Min	222	1105	61
Max	404	3569	91
Mean	304	2131	82
SD	63	626	7
n (experiments)		18

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	rel.viabilitymean[%]
Min	212	223	54
Max	428	1049	93
Mean	310	521	86
SD	60	223	9
n (experiments)		18

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In summary, after 24 hours of exposure to test substance Calcium disulphamate CD86 and CD54 expression was not induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance Calcium disulphamate does not induce dendritic cell activation.

Executive summary:

The potential of test substance Calcium disulphamate to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. No cytotoxicity was observed.

In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 experiments were performed.

In summary, after 24 hours of exposure to test substance Calcium disulphamate CD86 and CD54 expression was not induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance Calcium disulphamate does not induce dendritic cell activation.