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Diss Factsheets

Administrative data

Description of key information

DPRA (in chimico)

KeratinoSens (in vitro)

HUMAN-CELL LINE ACTIVATION TEST (h-CLAT)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September-October 2017
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:
other: in vitro Keratinosens
Justification for non-LLNA method:
The objective of the KeratinoSens assay is to evaluate the potential of the test item to activate the Nrf2 transcription factor. This test is part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non sensitizers in the context of an integrated approach to testing and assessment. It is an alternative method in order to avoid animal study
Specific details on test material used for the study:
Batch No.: . 42964
Storage conditions: . At room temperature. Protected from humidity.
Correction factor: . No correction factor
Expiry (or re-test) date: January 2019
Details on the study design:
Methods
The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed as part of this study.

PRINCIPLE OF THE ASSAY
This in vitro test uses Human adherent HaCaT keratinocytes, an immortalized cell line. The KeratinoSens is a stably transfected cell line with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence.
Potential skin sensitizers are applied to the cells at 12 different concentrations for a period of 48 hours. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The luciferase reporter gene is under control of a single copy of the ARE-element of the human AKR1C2. The luciferase production is measured by flash luminescence.
In parallel, cytotoxicity is measured by a MTT reduction and is taken into consideration in the interpretation of the sensitisation results. This evaluation is performed in at least two independent runs.
Positive control results:
Cinnamic Aldehyde (positive control item)
Gene induction values, Imax and EC1.5 values obtained with the positive control for each run:
run 1: Imax=6.71 and EC1.5=4.29
run 2: Imax=7.44 and EC1.5=8.22
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.27
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: IC50
Value:
6.67
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: IC30
Value:
5.47
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: 2
Parameter:
other: Imax
Value:
1.14
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 2
Parameter:
other: IC50
Value:
9.97
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: 2
Parameter:
other: IC30
Value:
8.1
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:
All acceptance criteria were fulfilled for the positive and negative controls in each run. Both runs were therefore considered to be valid.

KERATINOSENS RUN

The Imax, IC30, IC50, EC1.5 and viability values obtained for cells treated with test item in each run as well as the mean and SD values are presented in Appendix 2 of the report. The viability values (%), induction values, Imax, IC30, IC50 and EC1.5 values obtained with the positive control are presented in Appendix 3 of the report. In addition the luminescence values of all negative control wells and the %CV between these values for each run are also presented in Appendix 3 of the report.

All acceptance criteria were fulfilled for the positive and negative controls in each run. Both runs were therefore considered to be valid.

First run

This run was performed using the following concentrations: 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.

At these tested concentrations:

. a slight precipitate was observed at the highest concentration of 2000 μM at the end of the 48-hour treatment period,

. a high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 7.81 μM,

. the corresponding calculated IC30 and IC50 were 5.47 and 6.67 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was < 1.5 (i.e. 1.27).

The evaluation criteria for a negative response were met in this run.

Second run

Due to the cytotoxicity observed in the first run, a lower and narrower range of concentrations was used in the second run. This run was therefore performed using the following concentrations: 0.23, 0.32, 0.45, 0.64, 0.90, 1.3, 1.8, 2.5, 3.6, 5.0, 7.1, 10 μM in culture medium containing 1% DMSO.

At these tested concentrations:

. no precipitate was observed in any test item-treated wells at the end of the 48-hour treatment period,

. a decrease in cell viability (i.e. cell viability < 70%) was only noted at the highest concentration of 10 μM,

. the corresponding calculated IC30 and IC50 were 8.10 and 9.97 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was again < 1.5 (i.e. 1.14). The evaluation criteria for a negative response were also met in this run.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item HEXAMIDINE DIISETHIONATE, noted as highly cytotoxic at concentrations ≥ 10 μM, was considered as negative in the KeratinoSens assay up to the highest analyzable concentration of 7.1 μM. Therefore, the test item HEXAMIDINE DIISETHIONATE was considered to have no potential to activate the Nrf2 transcription factor, under the experimental conditions of this study.
Executive summary:

All acceptance criteria were fulfilled for the positive and negative controls in each run. Both runs were therefore considered to be valid.

First run

This run was performed using the following concentrations: 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.

At these tested concentrations:

. a slight precipitate was observed at the highest concentration of 2000 μM at the end of the 48-hour treatment period, in test item-treated wells,

. a high decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 7.81 μM,

. the corresponding calculated IC30 and IC50 were 5.47 and 6.67 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was < 1.5 (i.e. 1.27).

The evaluation criteria for a negative response were met in this run.

CiToxLAB France/Study No. 45434 TIK/HEXAMIDINE DIISETHIONATE/LA MESTA Chimie Fine

10/32

Second run

Due to the cytotoxicity observed in the first run, a lower and narrower range of concentrations was used in the second run. This run was therefore performed using the following concentrations 0.23, 0.32, 0.45, 0.64, 0.90, 1.27, 1.8, 2.5, 3.6, 5.0, 7.1, 10 μM in culture medium containing 1% DMSO.

At these tested concentrations:

. no precipitate was observed in any test item-treated wells at the end of the 48-hour treatment period,

. a decrease in cell viability (i.e. cell viability < 70%) was only noted at the highest concentration of 10 μM,

. the corresponding calculated IC30 and IC50 were 8.10 and 9.97 μM, respectively,

. no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control, at any tested concentrations. Moreover, the Imax value was again < 1.5 (i.e. 1.14).

The evaluation criteria for a negative response were also met in this run.

The evaluation criteria for a negative response were met in both runs, the final outcome is therefore negative. This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October-November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD guideline No. 442E
Version / remarks:
"In vitro skin sensitization: human Cell Line Activation Test (h CLAT)", 29 July 2016.
With the following exceptions:
 reactivity check was performed for each ATCC batch of cells and each working cell bank, and not each time frozen cells are thawed. Validation of cells reactivity was guaranteed in each run by running concurrently both positive controls (NiSO4 and DNCB) instead of only one (DNCB),
 according to the OECD guideline No. 442E, the first Dose Range Finding (DRF) assay should be performed at the maximum concentration of 1000 µg/mL before running another assay with a maximum concentration of 5000 µg/mL if no cytotoxicity is noted in the first assay. In the present study design, the first DRF assay was performed at 5000 µg/mL if allowed by solubility. A second DRF assay was performed in case no concentration leading to viability > 75% was obtained in the first DRF assay,
 when preparing cells for treatment, they were seeded between 0.1 and 0.2 x 106 cells/mL before incubating them for 48h to 72 hours (as noted in the DB-ALM protocol), instead of at 0.2 x 106 cells/mL for 48h incubation, or 0.1 x 106 cells/mL for 72h as noted in the OECD guideline.
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
Type of study:
other: human Cell Line Activation Test (h-CLAT),
Justification for non-LLNA method:
PRINCIPLE OF THE ASSAY
The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. It is an alternative method in order to avoid animal study.

Specific details on test material used for the study:
Batch No.: . 42964
Storage conditions: . At room temperature. Protected from humidity.
Correction factor: No correction factor
Expiry (or re-test) date: January 2019
Details on the study design:
Methods
A solubility assessment was first performed in 0.9% NaCl and DMSO to select the vehicle and highest concentration to be used for test item formulation preparations.

Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay in order to select sub-toxic concentrations for testing in the main test.
The skin sensitizing potential of the test item was then tested in the main test in both successive runs.
In each run, the test item formulations were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37°C, 5% CO2 in a humidified incubator. A set of control wells was also added in each plate to guarantee the validity of each run. At the end of the incubation period, cells from each well were distributed to three wells of 96-well plate: the first well was labeled with IgG1-FITC antibodies, the second one was labeled with CD86-FITC antibodies and the third one was labelled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination.

For each run, the Mean Fluoresence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. Corrected MFI value from the corresponding vehicle control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

TEST SYSTEM
Cells
The THP-1 is an immortalized human monocytic leukemia cell line derived from an acute monocytic leukemia patient. The THP-1 cell line is obtained from ATCC (Ref: TIB-202, American Type Culture Collection, Manassas, USA) by the intermediate of LGC Standards (Molsheim, France).
The THP-1 cells are stored in a cryoprotective medium in a liquid nitrogen container.
Cells were grown using general culture procedures. They were cultured in cRPMI medium and maintained in a humidified incubator set at 37°C, 5% CO2 and were not allowed to exceed a cell density of 1 x 106 cells/mL or more than 30 passages.
The culture medium (cRPMI) was composed of RPMI 1640 with 10% FBS, 0.05 mM 2 mercaptoethanol and with penicillin and streptomycin.
During cell culturing, cell viability was checked using trypan blue.

Cell culture for testing
For testing, THP-1 cells were seeded at a density between 0.1 x 106 cells/mL and 0.2 x 106 cells/mL, and pre-cultured in culture flasks for 48 hours to 72 hours, respectively. Cell did not exceed density of 1 x 106 cells/mL. On the day of testing, cells harvested from culture flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. Then, 500 µL of cells suspension were distributed into a 24 well flat bottom plate (i.e. 1 x 106 cells/well).

Reactivity check
Two weeks after thawing, a reactivity check was performed to qualify the cells of each working cell bank before testing. A reactivity check assay was performed by testing cell response after contact with Lactic Acid, DNCB and NiSO4 under an internal CiToxLAB France reference number.
Results from reactivity check tests are compiled in CiToxLAB France internal data, with negative and positive control data obtained during each test.
Positive control results:
2 positive controls: NiSO4 and DNCB
Run A
%viability:84.7 for NiSO4 and 63.6 for DNCB
Run B
%viability:79.1 for NiSO4 and 59.9 for DNCB
Key result
Run / experiment:
other: DRF1
Parameter:
other:
Remarks:
CV75 (µg/mL)
Value:
31.99
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: DRF2
Parameter:
other:
Remarks:
CV75 (µg/mL)
Value:
19.82
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:
the mean CV75 was 25.90 µg/mL
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, HEXAMIDINE DIISETHIONATE, was found to be negative in the h-CLAT.
Executive summary:

Solubility assessment

The test item was found soluble in DMSO at the concentration of 250 mg/mL.

  DRF RESULTS

The following results were obtainedin the first DRF test (i.e.DRF 1):

.            at post-treatment observation, no abnormalities such as precipitate/emulsion, but it was noted that few cells remained in the well after treatment at concentrations≥62.50 µg/mL,

.            flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75% at concentrations≥62.50 µg/mL. The corresponding CV75 value was 31.99 µg/mL.

 

The following results were obtainedin the second DRF test (i.e.DRF 2):

.            at post-treatment observation, no abnormalities such as precipitate/emulsion or cell morphology modification were observed at any tested concentrations,

.            flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75% at concentrations≥31.25 µg/mL. The corresponding CV75 value was 19.82 µg/mL.

 

Based on the results from both DRF runs, the mean CV75 was25.90 µg/mL, and the highest concentration tested in the main test was therefore31.80 µg/mL (i.e.1.2-fold the mean CV75).

Dose-Range Finding

The test item induced a decrease in cell viability < 75% in both DRF runs and the calculated mean CV75 value was: 25.90 µg/mL. The highest concentration tested in the main test was therefore 31.80 µg/mL (i.e.1.2-fold the mean CV75).

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2017
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))
GLP compliance:
yes
Type of study:
other: DPRA
Justification for non-LLNA method:
The objective of the study is to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.
The reactivity of the test item was evaluated in chemico : it is an alternative method in order to avoid animal study
Specific details on test material used for the study:
Batch No.: . 42964
Storage conditions: . At room temperature. Protected from humidity.
Correction factor: No correction factor
Expiry (or re-test) date: January 2019
Details on the study design:
Methods
The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.
Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

PRINCIPLE OF THE ASSAY
The assay determines the chemical reactivity of the test item to cysteine and lysine peptides, which is a unifying characteristic of most skin sensitizing chemicals (Gerberick et al. 2004, Gerberick et al. 2007).
Reactivity (% depletion) is determined following 24-hour contact between test item and peptide in acetonitrile at the ratios 1:10 cysteine:test item and 1:50 lysine:test item by liquid chromatography with Ultra-Violet detection. Complete details of the analysis process are described in CiToxLAB France analytical method procedure "A LC/UV analytical method for the determination of Direct Peptide Reactivity".
Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide area in the three relevant reference control C samples (in the appropriate solvent) by using the following formula:formula:
% depletion=[1-(Peptide Peak Area in Replicate Injection/Mean Peptide Peak Area in relevant Reference Control C samples)]*100
Then, the mean percent depletion of the three replicates was calculated for each peptide as well as the mean of the percent cysteine and percent lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean % depletion. Peak areas and peptide concentrations are presented in the report. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated and reported.
Positive control results:
The positive control was cinnamaldehyde.
Mean depletion rate (%) of Cinnamaldehyde =64.06 (High reactivity)
Key result
Run / experiment:
other: 1
Parameter:
other: %depletion
Remarks:
cysteine peptide
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: %depletion
Remarks:
cysteine peptide
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: %depletion
Remarks:
cysteine peptide
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: %depletion
Remarks:
Lysine peptide
Value:
3.71
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: %depletion
Remarks:
lysine peptide
Value:
4.56
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: %depletion
Remarks:
Lysine peptide
Value:
2.86
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / 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. The study was therefore considered to be valid.
Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item HEXAMIDINE DIISETHIONATE, was considered to no/minimal peptide reactivity, though with limitations due to its precipitation with the peptides.
The test item is considered negative in the DPRA assay.
Executive summary:

Results

The test item was dissolved at 100 mM in 1:1 mixture of acetonitrile:milli-Q water.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

. for the cysteine peptide, the mean depletion value was 0.00%,

. for the lysine peptide, the mean depletion value was 3.71%.

The mean of the percent cysteine and percent lysine depletions was equal to 1.85%. Accordingly, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

Since precipitate was observed at the end of the incubation with the peptides, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity cannot be drawn with sufficient

confidence.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

The test item Hexamidine diisethionate is considered negative in the DPRA assay.

The test item HEXAMIDINE DIISETHIONATE, noted as highly cytotoxic at concentrations ≥ 10 μM, was considered as negative in the KeratinoSens assay up to the highest analyzable concentration of 7.1 μM. Therefore, the test item HEXAMIDINE DIISETHIONATE was considered to have no potential to activate the Nrf2 transcription factor, under the experimental conditions of this study.

Under the experimental conditions of this study, the test item, HEXAMIDINE DIISETHIONATE, was found to be negative in the h-CLAT.