Mercaptamine

Endpoint summary

Description of key information

A DEREK assessment, DPRA assay and KeratinoSens^TMassay were performed in accordance withSection 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

The DEREK assessment yielded an alert for skin sensitization based on the presence of the thiol group and predicted an EC3 of 1.3% (LLNA). Mercaptamine was considered to be positive in the DPRA and classified in the “high reactivity class”. The KeratinoSensTM assay also yielded a positive result. Based on the positive in chemico/in vitro skin sensitization tests Mercaptamine should be classified as a skin sensitizer. DPRA data indicate a high protein reactivity and QSAR modelling predicted an EC3 of 1.3% (LLNA). Based on this information, it is considered justified to classify Mercaptamine as skin sensitiser Cat. 1A.

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

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:

direct peptide reactivity assay (DPRA)

Details on the study design:

PREPARATION OF TEST ITEM
No correction for the purity/composition of the test item was performed.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection
the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).

Test item stock solutions were prepared freshly for each reactivity assay.

For both the cysteine and lysine reactivity assay 13.26 mg of Mercaptamine was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1719 μL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
Any residual volumes were discarded.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC)
(Ac-RFAACAA-COOH) or synthetic peptides
containing lysine (SPCL) (Ac-RFAAKAA-COOH).
The molecular weight is 750.9 g/mol for SPCC and
775.9 g/mol for SPCL.

Rationale: Recommended test system in the international OECD guideline for DPRA studies.

EXPERIMENTAL DESIGN

Preparation of Solutions for Cysteine Reactivity Assay
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.

SPCC Calibration Curve
A SPCC calibration curve was prepared as described in the table 1 below.

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table 2 below.

Preparation of Solutions for Lysine Reactivity Assay
Synthetic Peptide Containing Lysine (SPCL) Stock Solution A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.

SPCL Calibration Curve
A SPCL peptide calibration curve was prepared as described in the table 3 below.

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table 4 below.

SAMPLE INCUBATIONS
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation times between placement of the samples in the autosampler and analysis of the first RCcysB- and RClysB-sample was 24 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.

Prior to HPLC-PDA analysis the samples were visually inspected for precipitation

HPLC-PDA ANALYSIS
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following system:
System 1 (used for Cysteine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
- LC Column oven 300 (Thermo Scientific)
- Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
- Column Oven #151006 (Grace, Worms, Germany)
- Surveyor PDA detector (Thermo Scientific)

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the
following formula:
Percent Peptide Depletion = [1 − ( Peptide Peak Area in Replicate Injection (at 220 nm)
Mean Peptide Peak Area in Reference Controls (at 220 nm))] × 100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using
the Cysteine 1:10 / Lysine 1:50 prediction model (see table 5 below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer

Run / experiment:

mean

Parameter:

other: SPCC Depletion

Value:

92.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

mean

Parameter:

other: SPCL Depletion

Value:

3.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 92.6% SPCC depletion while in the lysine reactivity assay the test item showed 3.7% SPCL depletion. The mean of the SPCC and SPCL depletion was 48.2% and as a result the test item was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, Mercaptamine was considered to be positive in the DPRA.

Table 6: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for Mercaptamine

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and     reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Mercaptamine	92.6%	±3.6%	3.7%	±0.3%	48.2%	Positive: High reactivity

SD = Standard Deviation.

Interpretation of results:

other: used in weight-of-evidence for classification and labelling

Conclusions:

In conclusion, this DPRA test is valid. Mercaptamine was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

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

Details on the study design:

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
A solubility test was performed. The test item was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 200 mM. The 100-fold dilution of the 200 mM DMSO stock in DMEM formed also a clear solution (2000 µM). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).

In the main experiments the test item was dissolved in DMSO at 200 mM. From this stock 11 spike solutions in DMSO were prepared (2- and 1.25-fold dilution series in the first and second experiment, respectively). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM and 2000, 1600, 1280, 1024, 819, 655, 524, 419, 336, 268, 215 and 172 µM in experiment 1 and 2, respectively (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

Test item concentrations were used within 2.5 hours after preparation

Any residual volumes were discarded.

PREPARATION OF THE POSITIVE CONTROL
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.7.1, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

PREPARATION OF THE SOLVENT CONTROL
The solvent control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

BLANK
On each plate three blank wells were tested (no cells and no treatment).

TEST SYSTEM
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

RATIONALE
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

CELL CULTURE
Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/ml).

Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 29 - 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 46.9 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

SUBCULTURING
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock.

EXPERIMENTAL DESIGN

Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was 18 in experiment 1 and 20 in experiment 2.

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about
48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 experiments were performed.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

DATA INTERPRETATION
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative (Figure 1):
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s
t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.52 and the EC1.5 82 µM
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.17 and the EC1.5 97 µM.

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

5.7

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

18.98

Vehicle 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 above the threshold of 1.5-fold in at least one concentration.
The EC1.5 of the positive control was between 5 and 125 µM (82 µM and 97 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.52-fold and 2.17-fold in experiment 1 and 2, respectively).
Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.3% and 9.4% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Table 1: Overview Luminescence Induction and Cell Viability of Mercaptamine in Experiment 1 and 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 luminescence	1.12	1.01	1.03	1.18	1.12	1.09	1.04	1.15	1.30	1.86***	2.46	5.70
Exp 1 viability (%)	108.2	108.2	109.1	99.9	96.3	89.2	87.4	85.0	81.7	73.8	48.8	10.5
Concentration (µM)	172	215	268	336	419	524	655	819	1024	1280	1600	2000
Exp 2 luminescence	1.67***	1.85***	2.00***	1.98***	2.19***	2.51***	2.90***	3.36***	6.27***	15.37***	8.04***	18.98
Exp 2 viability (%)	124.1	120.2	121.1	128.3	109.2	111.4	112.2	124.4	112.9	111.8	127.7	3.9

*** p<0.001 Student’s t test

 

Table 2: Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.12	1.25	1.15	1.44	1.63***	2.52***
Exp 1 viability (%)	92.2	96.5	98.9	90.0	105.1	86.1
Exp 2 luminescence	1.05	1.05	1.28	1.13	1.80***	2.17***
Exp 2 viability (%)	97.0	101.5	101.4	107.8	115.8	116.1

*** p<0.001 Student’s t test

 

Table 3: Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	339	5.70	576	976
Test item Experiment 2	<172	18.98	1786	1851
Pos Control Experiment 1	82	2.52	NA	NA
Pos Control Experiment 2	97	2.17	NA	NA

NA = Not applicable

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

based on weight-of-evidence report

Conclusions:

Mercaptamine is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QSAR prediction (see attached justification for details).

Qualifier:

no guideline followed

Principles of method if other than guideline:

QSAR calculation - DEREK NEXUS

GLP compliance:

no

Remarks:

not applicable for QSAR calculations

Remarks on result:

positive indication of skin sensitisation

Remarks:

prediction from QSAR calculation

Interpretation of results:

other: used a weight-of-evidence approach for classification and labelling

Conclusions:

DEREK NEXUS version 5.0.2 yielded an alert for Mercaptamine for skin sensitization based on the presence of the thiol group. Mercaptamine is predicted to be sensitizing to the skin (plausible).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

In conclusion, taking all existing and relevant data into account, there is sufficient information to meet the information requirement of Section 8.3 of Annex VII and Mercaptamine should be classified as skin sensitiser Cat 1A in accordance with the CLP regulation (EC1272/2008,as amended).