2-{3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl}ethyl hydrogen phosphate

Endpoint summary

Description of key information

QSAR prediction: negative

Skin sensitisation (OECD 442C): positive

Skin sensitisation (OECD 442D): positive

WoE conclusion from in vitro skin sensitisation battery: skin sensitising

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:

07 - 22 Apr 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))

Version / remarks:

adopted 05 Feb 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

TEST METHOD
The direct peptide reactivity assay (DPRA) is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, the protein reactivity. The reativity of the substance towards model synthetic peptides containing either lysine or cysteine is quantified. In the DPRA the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance is quantified. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 and 258 nm. Cysteine- and lysine peptide depletion values are then calculated and used in the prediction model, which assigns the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier of synthetic peptides: JPT Peptide Technologies GmbH
- Peptide stock solution preparation: Stock solutions of each peptide were prepared by dissolution of pre-weighed aliquots of the approprate peptide in appropriate buffer solution.
Cysteine-containing peptide: 18.48 mg cysteine was dissolved in 36.584 mL phosphate buffer (pH 7.5).
- Concentration: 0.667 mM
Lysine-containing peptide: 18.82 mg lysine was dissolved in 35.159 mL ammonium acetate buffer (pH 10.2).
- Concentration: 0.667 mM

VEHICLE CONTROL
- Substance: water
- Justification for selecting vehicle: the test substance was soluble in the vehicle.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Preparation: The positive control was prepared as 100 mM solution in acetonitrile.

CO-ELUTION CONTROL
- Co-elution controls were set up in parallel to sample preparation without respective peptide solution to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide.

REFERENCE CONTROLS
- Acetonitrile was used to verify the accuracy of the calibration curve for peptide quantification (Reference contol A) and to verify the stability of the respective peptide over the analysis time (Reference control B)
- Water and acetonitrile were used to verify that the solvents do not impact the percent peptide depletion (Reference controls C)

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: minimum of 24 ± 2 h

NUMBER OF REPLICATES
for each peptide triplicates were prepared for treatment substance and controls

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent 1200 Series, with Chemstation, Rev. B.04.01
- Analytical Column: Agilent Zorbax SB-C18, 3.5 µm, 100 x 2.1 mm
Pre-column: Phenomenex, AJO-4286, 4.0 x 2.0 mm
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Column temperature: 30 °C
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm for quantitation and 258 nm as indicator for co-elution
- Injection volume: 10 μL
- peptide standards: calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, and a buffer blank were measured in parallel with the test substance samples

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean peptide depletion of the positive control for the cysteine peptide was between 60.8% and 100% (74.90 %). The mean peptide depletion of the positive control for the lysine peptide was between 40.2% and 69.0% (54.93%).

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

52.81

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

74.90

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

In the lysine run co-elution of the test substance and the lysine peptide due to a shift of the lysine peptide peak was observed.

Other effects / acceptance of results:

OTHER EFFECTS:
In the lysine run a a co-elution of the test substance with the lysine peptide peak was observed. Since the reference control samples allowed clear identification of the lysine peptide and since the test item did not elute between 6.5 and 7.7 min, it was clear that the lysine peptide peak had shifted and that the peak area of that peak did only represent remaining lysine peptide. As all validity criteria were fulfilled the results of the lysine run was not considered having an influence on the quality or validity of the overall results.

ACCEPTANCE CRITERIA

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Table 1: Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD7	0.0000	0.0000	0.0000	0.0000
STD6	153.8068	0.0167	139.6547	0.0167
STD5	321.6256	0.0334	281.4845	0.0334
STD4	642.2243	0.0667	555.3300	0.0667
STD3	1318.5444	0.1335	1100.1805	0.1335
STD2	2593.4377	0.2670	2176.1533	0.2670
STD1	5075.1016	0.5340	4280.0898	0.5340

 

Table 2: Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1193.1283	0.1239	74.99	74.90	0.25	0.34
	1187.7434	0.1233	75.10
	1210.7732	0.1258	74.62
Test Item	2310.8784	0.2412	51.07	52.81	1.77	3.35
	2143.9973	0.2237	54.60
	2230.5615	0.2328	52.77

Table 3: Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1861.8433	0.2304	55.78	54.93	0.78	1.42
	1926.2322	0.2384	54.25
	1904.2300	0.2357	54.77
Test Item	n.a.*
	n.a.*
	n.a.*

* not applicable due to co-elution

Table 4: Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Table 5: Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

Table 6: Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	n.a.	n.a.	n.a.	52.81	Moderate Reactivity	sensitizer
Positive Control	64.92	High Reactivity	sensitizer	74.90	Moderate Reactivity	sensitizer

 

Interpretation of results:

other: skin sensitising potential based on the key event "protein reactivity"

Conclusions:

Under the conditions of the test, the test substance showed reactivity towards selected proteins. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

(Q)SAR prediction

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:

1. SOFTWARE
Lhasa Limited Derek Nexus

2. MODEL (incl. version number)
Derek Nexus: 5.0.1 and Nexus: 2.1.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Smiles Code: CC1=C(CCOP([O-])(O)=O)SC=[N+]1CC2=CN=C(C)N=C2N
CAS: 10023-48-0

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Structural alerts

5. APPLICABILITY DOMAIN
- Descriptor domain: The target chemical falls within the applicability domain of the prediction.

6. ADEQUACY OF THE RESULT
Within Derek, there are over 504 alerts covering a wide range of toxicological endpoints. An alert consists of a toxicophore (a substructure known or thought to be responsible for the toxicity) and is associated with literature references, comments and examples. The toxicity predictions are the result of two processes. The program first checks whether any alerts in the knowledge base match toxicophores in the query structure. The reasoning engine then assesses the likelihood of a structure being toxic. There are nine levels of confidence: certain, probable, plausible, equivocal, doubted, improbably, impossible, open, contradicted. The reasoning model considers the following information: a) the toxicological endpoint; b) the alerts that match toxicophores in the query structure; c) the physico-chemical property values calculated for the query structure; and d) the presence of an exact match between the query structure and a supporting example within the knowledge base.

Principles of method if other than guideline:

Calculation based on Derek Nexus: 5.0.1 and Nexus: 2.1.0. QSAR prediction of the skin sensitisation potential of the test substance.

GLP compliance:

no

Remarks on result:

no indication of skin sensitisation

No alerts were matched. Endpoints not firing any alerts at the selected reasoning level were occupational asthma, respiratory sensitisation, photoallergenicity and skin sensitisation.

Interpretation of results:

other: the prediction results was negative for skin sensitising potential, based on QSAR prediction (DEREK Nexus). The results may only be used for classification purposes together with other data

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 - 24 May 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)

Version / remarks:

adopted in Feb 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the study design:

TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance by addressing the second molecular key event of the Adverse Outcome Pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement compared with the respective solvent controls is used to support the discrimination between skin sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 6 (Experiment 1), 8 (Experiment 2)

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-pyruvate, 10% fetal bovine calf serum and 1% geneticin (final concentration 500 µg/mL)
Assay medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-g lucose and Na-pyruvate and 10% fetal bovine calf serum
Test substance exposure medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-Pyruvate and 1% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

TEST CONCENTRATIONS
0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000 and 2000 μM

CONTROLS
Solvent control:
- Substance: DMSO
Positive control:
- Substance: cinnamic aldehyde
- Final concentration: 4 - 64 μM

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 1 h
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 600 nm
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

DETERMINATION OF LUMINESCENCE
- Cell lysis reagent: Luciferase Cell Culture Lysis 5x Reagent Kit (Promega, Cat. No. E1531, Lot No. 000087405)
- Luciferase reagent: Luciferase Assay Kit (Promega, Cat. no. E1501, Lot. No. 0000237533)
- Device: plate reader

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.28 in experiment 1; 2.65 in experiment 2).

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: luciferase activity

Value:

3.76

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: cell viability [%]

Value:

79.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 [µM]

Value:

810.73

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: luciferase activity

Value:

2.75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: cell viability [%]

Value:

66

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5 [µM]

Value:

268.21

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: The average coefficient of variation of the luminescence reading for the vehicle control was < 20% (19.7% in and 12.5% in Experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.28 and 2.65 in Experiment 1 and 2, respectively) and the calculated EC1.5 value was between 7 and 34 µM (32.87 and 15.08 µM in Experiment 1 and 2, respectively).

In the first experiment, a max luciferase activity (Imax) induction of 3.76 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 79.6%. The further concentrations showed no significant luciferase induction >1.5. The calculated EC1.5 was < 1000 µM (810.73 µM).
In the second experiment, a max luciferase activity (Imax) induction of 2.75 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 66.0%. The lowest tested concentration with a significant luciferase induction >1.5 (1.70) was found to be 500 µM. The corresponding cell viability was >70% (71.9%). The calculated EC1.5 was < 1000 µM (268.21 µM).

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100.0	100.0	100.0	0.0
Positive Control	4.00	119.3	108.4	113.9	7.7
	8.00	105.9	120.5	113.2	10.3
	16.00	112.8	120.6	116.7	5.5
	32.00	121.9	126.1	124.0	3.0
	64.00	128.7	133.3	131.0	3.2
Test Item	0.98	92.1	92.4	92.3	0.2
	1.95	79.0	84.4	81.7	3.9
	3.91	78.7	85.2	81.9	4.6
	7.81	79.2	85.8	82.5	4.7
	15.63	79.1	84.9	82.0	4.1
	31.25	79.0	88.4	83.7	6.6
	62.50	78.6	86.6	82.6	5.7
	125.00	77.8	79.0	78.4	0.9
	250.00	80.2	86.0	83.1	4.1
	500.00	77.8	71.9	74.9	4.2
	1000.00	78.7	73.3	76.0	3.8
	2000.00	79.6	66.0	72.8	9.6

 

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.05	1.50	1.19	1.25	0.23
	8.00	1.38	1.20	1.30	1.29	0.09
	16.00	1.33	1.21	1.27	1.27	0.06
	32.00	1.43	1.52	1.32	1.42	0.10
	64.00	4.85	3.80	4.18	4.28	0.53	*
Test Item	0.98	1.23	0.80	1.07	1.03	0.22
	1.95	1.01	0.63	0.90	0.85	0.19
	3.91	0.97	0.69	0.98	0.88	0.16
	7.81	0.87	0.68	0.97	0.84	0.15
	15.63	0.78	0.71	0.97	0.82	0.13
	31.25	0.85	0.68	0.96	0.83	0.14
	62.50	0.87	0.71	0.99	0.86	0.14
	125.00	1.10	0.70	0.95	0.91	0.20
	250.00	1.01	0.81	1.25	1.02	0.22
	500.00	1.01	1.25	1.53	1.26	0.26
	1000.00	1.08	1.78	2.08	1.64	0.51
	2000.00	4.29	3.24	3.74	3.76	0.53	*

* = significant induction according to Student’s t-test, p<0.05

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.12	1.19	1.15	1.15	0.04
	8.00	1.10	1.24	1.27	1.21	0.09
	16.00	1.75	1.33	1.53	1.54	0.21	*
	32.00	1.87	1.70	1.96	1.85	0.13	*
	64.00	2.31	2.72	2.92	2.65	0.31	*
Test Item	0.98	1.15	1.11	1.18	1.15	0.04
	1.95	1.12	1.15	1.07	1.12	0.04
	3.91	1.04	1.05	1.14	1.08	0.06
	7.81	1.20	1.03	1.07	1.10	0.09
	15.63	1.06	1.07	1.38	1.17	0.18
	31.25	1.12	1.06	1.14	1.11	0.04
	62.50	1.22	1.13	1.19	1.18	0.04
	125.00	1.33	1.08	1.22	1.21	0.12
	250.00	1.42	1.46	1.58	1.48	0.08
	500.00	1.71	1.81	1.57	1.70	0.12	*
	1000.00	2.03	1.78	1.72	1.84	0.17	*
	2000.00	2.99	2.43	2.83	2.75	0.29	*

* = significant induction according to Student’s t-test, p<0.05

Table 4: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.25	1.15	1.20	0.06
	8.00	1.29	1.21	1.25	0.06
	16.00	1.27	1.54	1.40	0.19
	32.00	1.42	1.85	1.63	0.30
	64.00	4.28	2.65	3.46	1.15
Test Item	0.98	1.03	1.15	1.09	0.08
	1.95	0.85	1.12	0.98	0.19
	3.91	0.88	1.08	0.98	0.14
	7.81	0.84	1.10	0.97	0.18
	15.63	0.82	1.17	0.99	0.25
	31.25	0.83	1.11	0.97	0.19
	62.50	0.86	1.18	1.02	0.23
	125.00	0.91	1.21	1.06	0.21
	250.00	1.02	1.48	1.25	0.33
	500.00	1.26	1.70	1.48	0.31
	1000.00	1.64	1.84	1.74	0.14	*
	2000.00	3.76	2.75	3.25	0.71	*

* = significant induction according to Student’s t-test, p<0.05

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	810.73	268.21	539.47	383.62
Imax	3.76	2.75	3.25	0.71
IC30[µM]	n.a.	1455.58	1455.58	-
IC50[µM]	n.a.	n.a.	-	-

n.a. = not applicable

Interpretation of results:

other: skin sensitising potential based on the key event “inflammatory response in keratinocytes”

Conclusions:

Under the conditions of the test, the test substance has a keratinocyte activating potential. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Sensitisation

The skin sensitizing potential of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium (CAS 10023-48-0) was evaluated in a combination of non-animal methods (in silico, in chemico and in vitro).

 

(Q)SAR

The skin sensitisation potential of the test substance was predicted using a QSAR model Derek Nexus (5.0.1) and Nexus (2.1.0). A prediction of the potential for skin sensitisation, respiratory sensitisation, occupational asthma and photoallergenicity was performed. A prediction for a specific hazard potential is given as an alert, which consists of a toxicophore (a substructure known or thought to be responsible for the toxicity) and is associated with literature references, comments and examples. There were no alerts for any of the predictions performed.

 

In chemico/in vitro

The skin sensitising potential of the test substance was assessed in a Direct Peptide Reactivity Assay performed according to OECD guideline 442C and in compliance with GLP (reference 7.4.1-1). Test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. The mean depletion of the cysteine peptide was 52.81%. Using the prediction model defined in the guideline, the test substance is allocated to the reactivity class “moderate reactivity”. In the lysine run a co-elution of the test substance with the lysine peptide peak was observed. However, all validity criteria were fulfilled, and this shift was not considered having an influence on the quality or validity of the overall results. The positive control showed high reactivity towards the synthetic peptides as the mean depletion of both peptides was 64.92%.

Under the conditions of the test, the test substance showed reactivity towards the cysteine peptide. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

 

The activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test in the transgenic KeratinoSens™ cell line performed according to OECD guideline 442D and in compliance with GLP (reference 7.4.1-2). Cells were incubated with test substance concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000, 2000 µM for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. The study was conducted in two independent experiments. In the first experiment a max luciferase activity (Imax) induction of 3.76 was determined at a test substance concentration of 2000 µM. The corresponding cell viability was 79.6%. In the second experiment a max luciferase activity (Imax) induction of 2.75 was determined at a test substance concentration of 2000 µM. The corresponding cell viability was 66.0%. Therefore in both experiments the luciferase activity induction was >1.5. The lowest tested concentration with a significant luciferase induction >1.5 (1.70) was found to be 500 µM. The corresponding cell viability was >70% (71.9%). The EC1.5 was 810.73 µM and 268.21 µM in Experiment 1 and 2, respectively. Under the conditions of the test, the test substance has a keratinocyte activating potential.

Conclusion

A weight of evidence approach was applied to the available data on skins sensitisation, as the result of the QSAR prediction was negative, while the results of the 2 in chemico/in vitro tests were positive. The positive results of validated in chemico/in vitro tests covering 2 key events in the molecular initiating event leading to skin sensitisation are considered more reliable than the result of a QSAR prediction, which is based on (mainly structural) similarities between the test substance and known skin sensitisers. Therefore the test substance is considered to show a skin sensitising potential.

 

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance meet the classification criteria for Skin Sens 1, H317 according to Regulation (EC) 1272/2008.