N-(carbamoylmethyl)taurine

Endpoint summary

Description of key information

The in chemico skin sensitisation study conducted on the substance, which was incubated with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) for 22 h at 25 °C. The SPCC peptide depletion was obtained from experiment 4 and SPCL peptide depletion obtained from experiment 1, mean Percent Depletion Values were calculated as 1.30 %. In conclusion, the substance has ‘no or minimal reactivity’ according to Cystine 1:10 or Lysine 1:50 prediction model.

An in vitro study was performed to determine the potential of the substance to activate the Nrf2 transcription factor (sensitizing potential), employing the LuSens cell line. Two independent experiments were conducted, exposing the following concentrations 269 µM, 323 µM, 388 µM, 465 µM, 558 µM, 670 µM, 804 µM, 965 µM, 1157 µM, 1389 µM, 1667 µM, 2000 µM for 48 h. All substance concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction. In all tested concentrations of the substance no increase ≥ 1.5 fold in luciferase induction in comparison to the solvent control was measured. Therefore, the substance is considered to be negative for skin sensitising potential under the conditions of this study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th November 2018 - 20th March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 04. Feb. 2015

Deviations:

yes

Remarks:

The composition of the samples of the reference control C (water) in the Cys-peptide assay experiments 1, 2 and 3 was 750µL Peptide-Solution+250µL water. Considered uncritical by the test lab as experiments were repeated employing the correct composition.

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM (European Union Reference Laboratory for alternatives to animal test-ing): “DB-ALM Protocol n° 154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensi-tisation Testing.”

Version / remarks:

29th June 2015

Deviations:

yes

Remarks:

The composition of the samples of the reference control C (water) in the Cys-peptide assay experiments 1, 2 and 3 was 750µL Peptide-Solution+250µL water. Considered uncritical by the test lab as experiments were repeated employing the correct composition.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended OECD test for in vitro skin sensitisation potential.

Specific details on test material used for the study:

Batch no. GL865320171205
Appearance: White crystalline powder
Composition: 2-((2-Amino-2-oxoethyl)amino)ethanesulfonic acid
CAS No: 7365-82-4
EINECS-No: 230-908-4
Molecular formula: C4H10N2O4S
Molecular weight: 182.198 g/mol
Purity: ≥99 % Assay (Titration)
Homogeneity: homogeneous
Stability H2O: stable; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Solubility H2O: soluble; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Production date: 05. Dec. 2017
Expiry date: 05. Dec. 2019
Storage: Room Temperature (20 ± 5 °C)

Details on the study design:

The study was performed in order to evaluate the reactivity of the substance towards cysteine (Cys-) and lysine (Lys-) containing peptides. A substance solution in water and the respective peptide was incubated 22 h at 25 °C in the experiments 1, 2 and 4 and for 22h10min in experiment 3, respectively. The peptide concentration after the incubation was measured using HPLC-UV.
Three replicates were prepared using 1:10 and 1:50 molar ratio of the substance with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without substance were incubated and measured simultaneously.

Four experiments were performed.
Experiment 1 was not valid for the Cys-peptide assay, because the mean peptide concen-tration of the reference control C with water was not in the given range. For the Lys-peptide the assay in experiment 1 was valid and the results are reported here.
The second experiment was performed only for the Cys-peptide. It was invalid because of a tech-nical error during the HPLC measurement and thereby missing values for the positive control, the substance and both reference controls C.
In Experiment 3 again only the Cys-peptide assay was performed and the mean peptide concentration of the reference control C with water was not in the given range. Therefore, the experiment was not valid.
The fourth experiment was valid for the Cys-peptide and the results are reported here.
The invalid experiments are not reported in this report, but the raw data are kept in the test facility in the GLP- archive.

Test System.
Peptides with ≥95 % purity were used.

Cys-Peptide (Cysteine), RS-synthesis.
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Batch no.: P180711-LC180433
Purity: 97.89 %

Lys-Peptide (Lysine), GeneCust.
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Batch no.: P180418-LL107617
Purity: 97.62%

Controls:
Positive control.
Positive controls were treated identically as the substance. The following positive controls were used:
Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %, batch no. MKBT8955V, expira-tion date: Feb. 2020) was used as 100 mM (± 10 %) solution in acetonitrile for the Cys-peptide;
2,3-Butanedione (CAS 431-03-8, ≥99 %, batch no. BCBM5232V, expiration date: 29. Jan. 2019) was used as 100 mM (± 10 %) solution in acetonitrile for the Lys-peptide.
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide. Therefore, the acceptance criteria have to be adapted to the range of percent Lys-peptide depletion given in the proficiency section of the guideline OECD 442C. The mean peptide depletion value for the positive control 2,3-butanedione should be 10.0 % - 45.0 % with a maximum standard deviation < 11.6 % for the Lys-peptide.


Solvent controls.
For both peptides, four sets of solvent controls using acetonitrile instead of substance stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion. Additionally, a solvent control containing water instead of substance solution was prepared in triplicate (set C(water)) and also incubated and analysed together with the samples and was used for calculation of the depletion of the substance.

Co-elution control.
Sample prepared from the respective peptide buffer and the substance, but without peptide.

Analytical instrument.
6HPLC system
Designation: HPLC_4
Components: Degasser G1322A
Quaternary pump: G1311A
Autosampler: G1313A
Column compartment: G1316A
UV/VIS-Detector: DAD G1315A
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR15b Build 4981
Usage and calibration followed the corresponding SOP 114 00 526, edition 1 valid from 12. Dec. 2016.

Column
An ACE Excel SuperC18 150x3 mm column with 3 µm particles and pre-column Phenom-enex SecurityGuard C18, 4x3 mm was used. This column was selected because it delivers substantially better peak shape for the peptides than the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline.

HPLC program
Flow rate: 0.55 mL/min
Injection volume: 7 µL
Column temperature: 30 °C
Wavelength 1: 220 nm
Wavelength 2: 258 nm

Positive control results:

Cinnamaldehyde in acetonitrile (for the Cys-peptide) mean depletion (%) 81.91+/- 1.24.
2,3-Butanedione solution in acetonitrile (for the Lys-peptide) mean depletion (%) 27.27 +/-1.81.

Key result

Run / experiment:

other: 1

Parameter:

other: peptide depletion (%)

Remarks:

Cys-Peptide

Value:

0.73

Vehicle controls validity:

not applicable

Negative controls validity:

other: Solvent contol

Remarks:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: peptide depletion (%)

Remarks:

Cys-Peptide

Value:

2.94

Vehicle controls validity:

not applicable

Negative controls validity:

other: Solvent contol

Remarks:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 3

Parameter:

other: peptide depletion (%)

Remarks:

Cys-Peptide

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

other: Solvent contol

Remarks:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: peptide depletion (%)

Remarks:

Lys-Peptide

Value:

-0.42

Vehicle controls validity:

not applicable

Negative controls validity:

other: Solvent contol

Remarks:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: peptide depletion (%)

Remarks:

Lys-Peptide

Value:

-0.11

Vehicle controls validity:

not applicable

Negative controls validity:

other: Solvent contol

Remarks:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 3

Parameter:

other: peptide depletion (%)

Remarks:

Lys-Peptide

Value:

0.14

Vehicle controls validity:

not applicable

Negative controls validity:

other: Solvent contol

Remarks:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria:
a) The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100.0 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide;
b) The mean peptide depletion value for the positive control 2,3-butanedione should be 10.0 % - 45.0 % with a maximum standard deviation < 11.6 % for the Lys-peptide;
c) The standard deviation for the substance replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion.

Assessment:
a) The mean peptide depletion with a value of 81.91 % and the standard deviation of 1.24 % of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and < 14.9 %, respectively, for the Cys-peptide;
b) The mean peptide depletion with a value of 27.27 % and the standard deviation of 1.81 % of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and < 11.6 %, respectively, for the Lys-peptide;
c) The standard deviation for the substance replicates with 1.67 % was < 14.9 % for the percent cysteine depletion for the substance;
The standard deviation for the substance replicates with 0.08 % was < 11.6 % for the percent lysine depletion for the substance.

Interpretation of results:

GHS criteria not met

Conclusions:

In the DPRA study the substance is predicted to be “negative” with no or minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the substance possesses no or minimal skin sensitisation potential.

Executive summary:

The objective of this study was to determine the reactivity of the substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the substance with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the substance to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. The study was conducted in accordance with OECD TG 442C, 'In Chemico Skin Sensitization: Direct Peptide Reactivity Assay'.

Acetonitrile was found to be an appropriate solvent to dissolve the substance and was therefore used in this study.  The positive control used for the cysteine peptide was cinnamaldehyde. As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione was used as positive control showing mid-range depletion for the lysine peptide.The validation parameters were all within the acceptability criteria for the DPRA. The SPCC peptide depletion was obtained from experiment 4 and SPCL peptide depletion obtained from experiment 1. In the cysteine reactivity assay the substance showed 2.55% SPCC depletion while in the lysine reactivity assay the substance showed 0.05% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.30% and as a result the substance was considered to be negative in the DPRA and considered to be in the “no or minimal 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:

key study

Study period:

27th August 2018 - 13th September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD. (22. May 2015). PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS. ENV/JM/MONO(2015)6.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735 adopted 14. Feb. 2017: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Batch no. GL865320171205
Appearance White crystalline powder
Composition 2-((2-Amino-2-oxoethyl)amino)ethanesulfonic acid
Purity >=99% Assay (Titration)
Homogeneity homogeneous
Expiry date 05. Dec. 2019
Storage Room temperature (20 ± 5 °C)

Details on the study design:

Reasons for the Choice of the LuSens Cell Line
The LuSens cell line was specially designed for this test system by the BASF SE (Ludwigshafen, Germany). It employs the use of a luciferase reporter gene placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).

Cell Culture
The LuSens cell line was obtained from the BASF SE (Ludwigshafen, Germany). For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells (mycoplasma contami-nation free), which guarantees similar parameters of the experiment and reproducible characteristics of the cells.

For the Cytotoxicity Range Finder Assay cells of passage 10 were used. For the main experiments cells of passage 12 and 14 were used. After thawing the cells were cultivated in DMEM (9 % FCS (Fetal calf serum)) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Positive control results:

Range finding study: Relative viability = 81.7 % (standard deviation= 1.62 %).
Experiment I: Relative viability = 77.0 % (standard deviation= 3.23 %);
Luciferase induction = 4.1 fold (standard deviation= 3.48 %).
Experiment II: Relative viability = 78.4 % (standard deviation= 1.70 %);
Luciferase induction = 4.0 fold (standard deviation= 3.20 %).

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Luciferase induction

Remarks:

(fold in comparison to the solvent control)

Value:

0.9

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: Experiment 2

Parameter:

other: luciferase induction

Remarks:

(fold in comparison to the solvent control)

Value:

0.9

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:

No considerable reduction of the viability was detected (all values ≥ 77 %). No cytotoxic effect was observed at all substance concentrations. The viability values were all > 91 % or > 96 % in the first and second experiment, respectively, and therefore analysable for luciferase induction.

Dose selection experiment. Results of Relative Viability [%] in CRFT.

Parameter	Concentration	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	[%]		[%]
Solvent Control	-	100.0	1.93	1.93
Growth Control	-	100.3	1.13	1.13
Negative Control	5000	98.2	1.47	1.50
Positive Control	80	81.7	1.32	1.62
Test item	0.98	102.1	1.47	1.44
Test item	1.95	98.1	0.75	0.76
Test item	3.91	100.3	2.01	2.00
Test item	7.81	98.8	3.62	3.67
Test item	15.63	98.0	4.98	5.08
Test item	31.25	96.5	7.67	7.95
Test item	62.5	98.1	3.37	3.43
Test item	125	100.3	1.71	1.71
Test item	250	102.7	0.24	0.23
Test item	500	102.3	1.07	1.04
Test item	1000	101.7	1.00	0.98
Test item	2000	103.6	0.83	0.81

No cytotoxic effect was observed at the controls as well as at all substance concentrations. The viability values were all above 81 %.

The test was valid and could be used for the determination of the concentrations to be tested in the main experiments.

The CV75 value could not be calculated because of no cytotoxicity.

Results of experiment I

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.07	7.42	100.0	2.32	2.32
Growth Control	-	1.0	0.08	8.41	101.3	2.49	2.45
Negative Control	5000	1.0	0.11	11.16	99.1	2.50	2.52
Positive Control	80	4.1	0.14	3.48	77.0	2.49	3.23
Test item	269	1.0	0.10	10.90	99.0	0.92	0.93
Test item	323	1.0	0.07	6.82	98.5	3.77	3.83
Test item	388	1.0	0.11	11.00	95.6	0.59	0.62
Test item	465	1.0	0.08	7.98	96.6	1.01	1.04
Test item	558	1.0	0.05	4.85	91.6	2.34	2.56
Test item	670	1.0	0.10	9.89	96.5	0.82	0.85
Test item	804	1.0	0.05	5.20	99.0	4.51	4.56
Test item	965	1.0	0.08	8.07	98.8	0.79	0.80
Test item	1157	0.9	0.03	3.52	99.3	0.54	0.54
Test item	1389	0.9	0.05	5.42	97.3	1.71	1.76
Test item	1667	0.9	0.09	10.17	98.2	3.08	3.14
Test item	2000	1.0	0.07	7.14	98.3	1.38	1.41

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 77 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.0 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 4.1 fold in comparison to the solvent control.

No cytotoxic effect was observed at all substance concentrations. The viability values were all > 91 % and therefore analysable for Luciferase induction.

In the Luciferase assay, none of the tested concentrations induced a Luciferase induction above or equal 1.5 fold in comparison to the solvent control.

Results of experiment II

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.07	7.42	100.0	2.32	2.32
Growth Control	-	1.0	0.08	8.41	101.3	2.49	2.45
Negative Control	5000	1.0	0.11	11.16	99.1	2.50	2.52
Positive Control	80	4.1	0.14	3.48	77.0	2.49	3.23
Test item	269	1.0	0.10	10.90	99.0	0.92	0.93
Test item	323	1.0	0.07	6.82	98.5	3.77	3.83
Test item	388	1.0	0.11	11.00	95.6	0.59	0.62
Test item	465	1.0	0.08	7.98	96.6	1.01	1.04
Test item	558	1.0	0.05	4.85	91.6	2.34	2.56
Test item	670	1.0	0.10	9.89	96.5	0.82	0.85
Test item	804	1.0	0.05	5.20	99.0	4.51	4.56
Test item	965	1.0	0.08	8.07	98.8	0.79	0.80
Test item	1157	0.9	0.03	3.52	99.3	0.54	0.54
Test item	1389	0.9	0.05	5.42	97.3	1.71	1.76
Test item	1667	0.9	0.09	10.17	98.2	3.08	3.14
Test item	2000	1.0	0.07	7.14	98.3	1.38	1.41

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 77 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.0 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 4.1 fold in comparison to the solvent control.

No cytotoxic effect was observed at all substance concentrations. The viability values were all > 91 % and therefore analysable for Luciferase induction.

In the Luciferase assay, none of the tested concentrations induced a Luciferase induction above or equal 1.5 fold in comparison to the solvent control.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test substance was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Executive summary:

An in vitro study was performed to investigate the potential of the substance to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.

The assay was performed in two independent experiments. Twelve concentrations of the substance were evaluated. The exposure time was 48 h. The following nominal concentrations of the substance were investigated in experiment I and II: 269 µM, 323 µM, 388 µM, 465 µM, 558 µM, 670 µM, 804 µM, 965 µM, 1157 µM, 1389 µM, 1667 µM, 2000 µM.

Precipitation of the substance was not visible up to the highest concentration. p-Phenylenediamine (80 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control. DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control. The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.

Since all acceptability criteria of the assay were met the study is valid.

 

No cytotoxic effect was observed in all tested substance concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction.

 

All substance concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction. In all tested concentrations of the substance no increase ≥ 1.5 fold in luciferase induction in comparison to the solvent control was measured. Therefore, the substance is considered to be negative for skin sensitising potential under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

On the basis of a negative, reliable, in vitro and in chemico studies performed on the substance, classification of the substance is not justified.