2-ethyl-3-methylpyrazine

Endpoint summary

Description of key information

The test substance does not activate keratinocytes (OECD 442D) and dendritic cells (OECD 442E). Therefore the test substance is considered not to be a skin sensitiser.

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:

weight of evidence

Study period:

22 February 2017 - 06 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

2016

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

Principles of method if other than guideline:

In addition, the h-CLAT was performed according to the methods described in the following publications:
- Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol In Vitro. 2012 Oct;26(7):1150-60.
- Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. (2010). A comparative evaluation of in vitro skin sensitization tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010 Aug;38(4):275-84.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Skin sensitisation (In vitro test system)
Cell line used: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202.

Technical material and conditions:
- Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 µg/mL of penicillin and 100 μg/mL of streptomycin).
- FACS buffer: PBS with 0.1% (w/v) BSA
- Antibodies: Anti - CD86 antibody (Clone: Fun-1), Anti – CD54 antibody (Clone: 6.5B5),
FITC labelled-mouse IgG1 (isotype control)

Controls used:
- Vehicle control: DMSO (final concentration 0.2%)
- Positive control: DNCB in DMSO (diluted with culture medium to a final concentration of 2 and 3 μg/mL DNCB)
- Isotype control: mouse IgG1.

Test procedure:

Dose Finding Assay, XTT Test:
Two independent cytotoxicity experiments were performed on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). All dose groups (eight concentrations) were tested in 7 replicates for each XTT test. The incubation period was 24 ± 0.5 hours. After incubation with the XTT labelling mixture the absorbance was measured at 450 nm.

Human Cell Line Activation Test (h-CLAT):
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining . The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry.

Acceptance criteria:
- Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
- In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.

Evaluation of results:
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE.

Positive control results:

The results of the positive control were within the historical control range.

Run / experiment:

other: First run_502 µg/ml

Parameter:

other: RFI % (CD54)

Value:

209.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: First run_242 µg/ml

Parameter:

other: RFI % (CD86)

Value:

178.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: First run_290 µg/ml

Parameter:

other: RFI % (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: First run_348 µg/ml

Parameter:

other: RFI % (CD86)

Value:

182.9

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: First run_418 µg/ml

Parameter:

other: RFI % (CD86)

Value:

158.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: First run_502 µg/ml

Parameter:

other: RFI % (CD86)

Value:

240

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: First run_602 µg/ml

Parameter:

other: RFI % (CD86)

Value:

233.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Second run repetition_all concentrations

Parameter:

other: RFI % (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Second run repetition_all concentrations

Parameter:

other: RFI % (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Third run_all concentrations

Parameter:

other: RFI % (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Third run_all concenctrations

Parameter:

other: RFI % (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

The results of the second run were not valid (cell viability < 90% at the highest tested test item concentration with a negative result). Therefore the second run was repeated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not applicable
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Results of the first h-CLAT run for the Test Item

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	230.7*	291.1*	75.0
	3.0	245.9*	279.9*	76.6
Test Item	168	130.0	141.4	105.0
	202	132.2	148.1	107.2
	242	153.0	178.1*	105.9
	290	142.2	150.0*	96.3
	348	153.0	182.9*	96.9
	418	184.8	158.1*	96.4
	502	209.6*	240.0*	99.7
	602	191.7	233.3*	89.0

 * RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥ 150% and CD54 ≥ 200%)

Table 2: Results of the second h-CLAT run repetition for the Test Item

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	400.9*	652.3*	56.9
	3.0	579.4*	564.5*	58.7
Test Item	168	100.0	84.8	102.1
	202	105.9	63.4	99.0
	242	119.8	81.4	100.9
	290	128.7	88.3	96.8
	348	102.0	50.3	93.1
	418	179.2	72.4	92.4
	502	148.5	117.9	99.4
	602	158.4	92.4	78.4

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥ 150% and CD54 ≥ 200%)

Table 2: Results of the third h-CLAT run for the Test Item

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	333.0*	592.5*	68.3
	3.0	342.7*	656.6*	67.4
Test Item	168	93.6	59.2	97.0
	202	90.4	69.6	97.7
	242	91.5	72.8	95.4
	290	193.6	96.0	102.6
	348	110.6	88.8	98.1
	418	128.7	64.8	97.4
	502	133.0	92.0	83.6
	602	158.5	116.8	80.3

 * RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥ 150% and CD54 ≥ 200%)

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not activate THP-1 cells up to the highest tested test item concentration (602 μg/mL). Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h- CLAT). The highest test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 168, 202, 242, 290, 348, 418, 502 and 602μg/mL.

The test substance was administered to THP-1 cells for 24 ± 0.5 hours. The test item was tested in 3 independent valid runs. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in at least 2 of 3 independent run data. Two out of three runs were NEGATIVE, therefore the test item is considered to have no skin sensitisation potential. The controls confirmed the validity of the study.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 March 2017 - 26 June 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:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system)
Cell line used: KeratinoSensTM (Givaudan, Switzerland)

Technical material and conditions:
- Maintenance Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS + 1 % Geneticin (final concentration: 500 µg/mL)
- Assay Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS
- Test Item Exposure Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 1 % FBS
- Luciferace reagent : Luciferase Assay Substrate (Promega, Cat. No.: E1501)
- Assay Buffer: Luciferase Assay Buffer (Promega, Cat. No.: E1501)
- Lysisbuffer: Luciferase Cell Culture Lysis (Promega Cat. No.: E1531)
- MTT Solution: MTT stock solution: 5 mg/mL MTT in DPBS
- SDS solution: 10% (wlv) SDS in dist. water
- DPBS solution: DPBS solution (without Ca2+/Mg2+)

Controls used:
- Vehicle control: DMSO: 1% (vlv) in test item exposure medium
- Positive control: Cinnamic aldehyde in DMSO, 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
- Blank control: vehicle control without cells

Test procedure:
Each concentration step of the test item (twelve concentrations) and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates.
The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a plate reader.
Cell viability was determined by a MTT assay. The test substance was incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 10% SDS solution the plate was incubated at 37 °C ± 1 °C and 5% CO2 overnight. The OD was measured at A = 600 nm.

Acceptance criteria:
- The luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations.
- The average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8.
- The EC1.5 value of the positive control is within two standard deviations of the historical mean.
- The average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Evaluation of results:
The test item is considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.

Positive control results:

The results of the positive control were within the historical control range.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.50

Remarks:

(the concentration resulting in a 1.50-fold luciferase induction)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No significant luciferase induction > 1.5 was found.

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.50

Remarks:

(the concentration resulting in a 1.50-fold luciferase induction)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No significant luciferase induction > 1.5 was found.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Remarks:

(maximal fold induction of luciferase)

Value:

1.05

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Remarks:

(maximal fold induction of luciferase)

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not applicable
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: 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	1.19	1.07	1.07	1.11	0.07
	8.00	1.19	1.05	1.05	1.09	0.08
	16.00	1.62	1.18	1.36	1.39	0.22
	32.00	1.96	1.45	1.68	1.70	0.26	*
	64.00	2.75	2.09	2.48	2.44	0.33	*
Test Item	0.98	0.92	1.01	0.95	0.96	0.04
	1.95	0.91	1.10	1.02	1.01	0.10
	3.91	1.11	1.06	0.89	1.02	0.11
	7.81	0.92	1.02	0.93	0.96	0.06
	15.63	1.00	0.80	1.04	0.95	0.13
	31.25	0.92	0.93	0.81	0.89	0.07
	62.50	0.99	0.89	0.87	0.92	0.07
	125.00	0.99	0.91	1.00	0.97	0.05
	250.00	0.97	0.87	0.90	0.92	0.05
	500.00	0.96	0.87	0.81	0.88	0.07
	1000.00	1.02	0.94	0.94	0.97	0.05
	2000.00	1.17	1.11	0.87	1.05	0.16

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

 

Table 2: 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	1.04	1.09	1.05	1.06	0.03
	8.00	1.13	1.24	0.97	1.11	0.13
	16.00	1.18	1.34	1.18	1.23	0.09
	32.00	1.67	2.26	2.12	2.02	0.31	*
	64.00	3.68	4.04	3.98	3.90	0.19	*
Test Item	0.98	0.96	1.08	0.76	0.93	0.16
	1.95	1.00	1.69	0.68	1.12	0.52
	3.91	0.84	1.17	0.79	0.93	0.21
	7.81	0.91	1.06	0.66	0.88	0.20
	15.63	0.79	1.07	0.88	0.92	0.14
	31.25	0.82	0.94	0.68	0.81	0.13
	62.50	0.84	0.93	0.69	0.82	0.12
	125.00	0.84	1.03	0.74	0.87	0.15
	250.00	0.78	0.92	0.69	0.80	0.12
	500.00	0.86	1.01	0.58	0.82	0.22
	1000.00	0.67	0.82	0.69	0.73	0.08
	2000.00	0.71	1.15	0.72	0.86	0.25

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

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSensTM cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The keratinocyte activating potential of test substance was evaluated in the LuSens Assay (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM in DMSO). Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.05 was determined. No significant luciferase induction >1.5 was found and no cytotoxic effect was observed within the tested concentration range. Therefore, no EC1.5 could be calculated. In the second experiment, a max luciferase activity (Imax) induction of 1.12 was determined. No significant luciferase induction >1.5 was found and no cytotoxic effect was observed within the tested concentration range. Therefore, no EC1.5 could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is considered as non sensitiser. The controls confirmed the validity of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a test battery of non in vivo experiments was performed, as a single in vitro/in chemico test is not sufficient for adequate judgment. Together the experiments cover all 3 key events of skin sensitisation. The protein reactivity is addressed with the Direct Peptide Reactivity Assay (DPRA). The Keratinocyte Activation Assay LuSens assay evaluates keratinocyte activation and the Human Cell Line Activation Test (h- CLAT) determines activation of dendritic cells. An evaluation of all tests in a Weight of Evidence approach is used to reach a final conclusion on the skin sensitisation potential of the test substance and its need for classification.

 

LuSens

The keratinocyte activating potential of test substance was evaluated in the LuSens Assay (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM in DMSO). Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.05 was determined. No significant luciferase induction >1.5 was found and no cytotoxic effect was observed within the tested concentration range. Therefore, no EC1.5 could be calculated. In the second experiment, a max luciferase activity (Imax) induction of 1.12 was determined. No significant luciferase induction >1.5 was found and no cytotoxic effect was observed within the tested concentration range. Therefore, no EC1.5 could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is considered as non sensitiser. The controls confirmed the validity of the study.

 

 

h-CLAT

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h- CLAT). The highest test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 168, 202, 242, 290, 348, 418, 502 and 602μg/mL.

The test substance was administered to THP-1 cells for 24 ± 0.5 hours. The test item was tested in 3 independent valid runs. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in at least 2 of 3 independent run data. Two out of three runs were NEGATIVE, therefore the test item is considered to have no skin sensitisation potential. The controls confirmed the validity of the study.

 

DPRA

As the LuSens Assay and the Human Cell Line Activation Test (h- CLAT) have shown no indication of skin sensitization a Direct Peptide Reactivity Assay (DPRA) was not performed.

 

Conclusion

The LuSens assay showed no activation of keratinocytes and the Human Cell Line Activation Test (h- CLAT) showed no activation of dendritic cells. As those two tests unanimously showed no indication of skin sensitization an additional assay addressing the third key event was not required and the test item is considered not to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776. As a result the substance is considered not to be classified for skin sensitisation.