p-cymene

Endpoint summary

Description of key information

Two key events of the Adverse Outcome Pathway (AOP) for skin sensitisation were investigated in a Direct Peptide Reactivity Assay (DPRA, OECD guideline 442C) and in an ARE-Nrf2 Luciferase Test (OECD guideline 442D) after treatment with the test item. The test item is considered not suitable for DPRA due to poor water solubility under the conditions of this study. Thus, this study was stopped after pre-experiments. In the ARE-Nrf2 Luciferase Test, the test substance did not activate the LuSens cells up to a concentration of 166 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP. Using Derek Nexus v. 6.0.1, no skin sensitising properties of the test item were estimated. As the substance is within the applicability domain of the model, the estimation can be regarded as accurate. In summary, it can be concluded that the test substance has no sensitizing properties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

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:

Please refer to the QMRF and QPRF files provided under the section attached justification.

Qualifier:

no guideline available

Principles of method if other than guideline:

Estimates the skin sensitising properties of chemicals using structural alert relationships.

GLP compliance:

no

Key result

Parameter:

other: alerts

Remarks on result:

no indication of skin sensitisation

Remarks:

QSAR predicted value. The substance is (not) within the applicability domain of the model.

Interpretation of results:

other: No prediction of skin sensitisation

Conclusions:

Using Derek Nexus v. 6.0.1, no skin sensitising properties of the test item were estimated. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

Executive summary:

The skin sensitising properties were estimated using Derek Nexus v. 6.0.1. No skin sensitising properties were estimated based on the described QSAR method (Derek, 2020).

 

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

 

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-06-30 to 2020-08-27

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:

06-2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

CONTROLS
- Medium control: Treatment medium
- Solvent: DMSO (final concentration 1% (v/v) in Treatment Medium), purity: ≥ 99%
- Positive control: EGDMA (final concentration 120 μM), CAS 97-90-5, purity: ≥ 97.5%
- Negative control: Lactic acid (final concentration 5000 μM), CAS 50-21-5, purity: ~ 90%

TEST ITEM PREPARATION
- The test item was dissolved or stably dispersed/suspended in DMSO to prepare a stock solution with a concentration of 200 mM immediately before treatment

- Test concentrations:
Dose finding assay (MTT assay): 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM
Main experiments: 66.7, 80.1, 96.1, 115, 138 and 166 µM

CELL CULTURE
- Incubation conditions: at 37°C ± 1.5°C and 5 ± 0.5% CO2
- Passage numbers: Passage 11 in the cytotoxicity test and 13 in the LuSens test for the main experiments 1 and 2, respectively
- Culture medium:
Cultivation medium: DMEM culture medium with Penicillin/Streptomycin (1% v/v), FBS (10% v/v) and puromycin (0.005% v/v)
Seeding Medium: DMEM culture medium with FBS (10% v/v)
Treatment Medium: DMEM culture medium with FBS (1% v/v)
- Seeding: 100 μL cell suspension (9000-11000 cells/well) in seeding medium, incubation for 24 hours ± 30 minutes

EXPERIMENTAL PROCEDURE
Dose finding test (MTT assay):
- MTT solution: MTT stock solution (5mg/mL in DPBS) in Treatment medium (1:10 soultion)
- Treatment: 150 μL treatment medium was added per well and 50 μL of the test item dilutions, the solvent, negative and positive controls and the medium control were added to the wells, respectively. At the end of the incubation period of 48 ± 1 hours, the cell cultures were microscopically evaluated for morphological alterations, precipitation or phase separation.
- Measurement of cell viability: At the end of the incubation period, cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the MTT working solution were added to each treatment well and the cells were incubated for 3 hours ± 30 minutes. After rinsing the MTT working solution, the cells of each well were treated with 100 μL MTT lysis agent (Isopropanol with
0.04 N HCl) for at least 30 minutes, while gently shaking. Thereafter the microplate was transferred to a microplate reader equipped with a 570 nm filter to measure the absorbance (reference wavelength 690 nm).

Main experiments (LuSens and MTT assay):
- Replicates: 2
- Treatment: For each main experiment one 96 well microtiter plate was prepared for MTT assay and one for the luciferase activity measurement.150 μL of treatment medium was distributed in each well. Thereafter, 50 μL of the test item and control dilutions and the medium control were added into the corresponding wells. At the end of the incubation period of 48 ± 1 hours, the cell cultures
were microscopically evaluated for morphological alterations, precipitation or phase separation.
- Measurement of cell viability (MTT): As described for the dose finding test
- Measurement of the Luciferase activity: The Steady-Glo® Mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® Substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part of Steady-Glo®-Mix. At the end of the incubation period, the Treatment Medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 seconds per well.

REASON FOR CHOICE OF SYSTEM
The LuSens cell-line is an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The plasmid contains a luciferase gene (reporter gene) which is under transcriptional control of an antioxidant response element (ARE) of the rat NQO1 gene. Genes dependent on the ARE such as NQO1 are known to be upregulated by contact sensitisers (OECD 442C).

Positive control results:

The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (Main experiment 1: 5.05; Main experiment 2: 4.69).

The positive control had a relative cell viability ≥ 70% as compared to the solvent control
(ME 1: 70.17%; ME 2: 90.97%).

Key result

Run / experiment:

other: Main experiment 2

Parameter:

other: x-fold luciferase induction

Value:

0.42

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: Main experiment 1

Parameter:

other: x-fold luciferase induction

Value:

1.32

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The average luciferase activity induction were not statistically significant.

Key result

Run / experiment:

other: Dose finding assay (cytotoxicity test)

Parameter:

other: CV75 value

Value:

138.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The CV75 value of the cytotoxicity test was calculated as 138.1 μM.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Results of the dose finding assay (cytotoxicity test)

                 

Treatment Group	Concentration		Absorbance (OD570)			Mean OD570	SD OD570	Mean OD570 blank corr.	Cell viability [%]
			Well 1	Well 2	Well 3
Blank			0.021
Solvent control						0.473	0.03	0.452	100.0
Medium control						0.574	0.07	0.553	122.25
Positive Control	120 uM		0.475	0.479		0.477	0.00	0.456	100.87
Negative Control	5000 uM		0.414	0.440	0.427	0.427	0.01	0.406	89.81
Test Item	C1	0.98 uM	0.305	0.411	0.405	0.374	0.06	0.353	78.01
	C2	1.95 uM	0.329	0.349	0.411	0.363	0.04	0.342	75.65
	C3	3.91 uM	0.403	0.426	0.441	0.423	0.02	0.402	89.00
	C4	7.81 uM	0.417	0.421	0.436	0.425	0.01	0.404	89.29
	C5	15.6 uM	0.432	0.439	0.429	0.433	0.01	0.412	91.21
	C6	31.3 uM	0.401	0.439	0.434	0.425	0.02	0.404	89.29
	C7	62.5 uM	0.353	0.388	0.409	0.383	0.03	0.362	80.15
	C8	125 uM	0.359	0.413	0.408	0.393	0.03	0.372	82.36
	C9	250 uM	0.018	0.065	0.142	0.075	0.06	0.054	11.94
	C10	500 uM	0.017	0.017	0.018	0.017	0.00	-0.004	0.00*
	C11	1000 uM	0.018	0.018	0.018	0.018	0.00	-0.003	0.00*
	C12	2000 uM	0.018	0.018	0.019	0.018	0.00	-0.003	0.00*

The CV75 value of the cytotoxicity test was calculated as 138.1 µM

 

 

Table 2: Results of the main experiment 1 (cell viability)

           

Treatment Group	Concentration		Absorbance (OD570)						Mean OD570	SD OD570	Mean OD570 blank corr.	Cell viability [%]
			Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank			0.015
Solvent control									0.264	0.05	0.249	100.0
Medium control									0.278	0.03	0.263	105.40
Positive Control	120 uM		0.157	0.213	0.176	0.201	0.202		0.190	0.02	0.175	70.17
Negative Control	5000 uM		0.171	0.179	0.207	0.207	0.195	0.191	0.192	0.01	0.177	70.91
Test Item	C1	66.7 uM	0.248	0.228	0.237				0.238	0.01	0.223	89.38
	C2	80.1 uM	0.253	0.277	0.227				0.252	0.03	0.237	95.27
	C3	96.1 uM	0.335	0.323	0.306				0.321	0.01	0.306	122.96
	C4	115.0 uM	0.288	0.271	0.235				0.265	0.03	0.250	100.22
	C5	138.0 uM	0.258	0.260	0.264				0.261	0.00	0.246	98.61
	C6	166.0 uM	0.062	0.139	0.250				0.150	0.09	0.135	54.32

 

 

Table 3: Results of the main experiment 1 (fold induction)

           

Treatment Group	Concentration		Luminescence						Mean Luminescence	SD Luminescence	Mean Luminescence blank corr.	Fold Induction
			Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank			155
Solvent control									211.2	11.47	56.2	1.00
Medium control									217.4	17.16	62.4	1.11
Positive Control	120 uM		473	451	436	414	421		439.0	23.76	284.0	5.05
Negative Control	5000 uM		214	207	222	214	222	236	219.2	10.01	64.2	1.14
Test Item	C1	66.7 uM	399	229	214				280.7	102.75	125.7	2.24
	C2	80.1 uM	347	251	229				275.7	62.75	120.7	2.15
	C3	96.1 uM	288	214	207				236.3	44.88	81.3	1.45
	C4	115.0 uM	259	251	200				236.7	32.01	81.7	1.45
	C5	138.0 uM	281	259	244				261.3	18.61	106.3	1.89
	C6	166.0 uM	222	236	229				229.0	7.00	74.0	1.32

The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was 5.4% in the main experiment 1

 

 

Table 4: Results of the main experiment 2 (cell viability)

           

Treatment Group	Concentration		Absorbance (OD570)						Mean OD570	SD OD570	Mean OD570 blank corr.	Cell viability [%]
			Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank			0.021
Solvent control									0.452	0.06	0.431	100.0
Medium control									0.374	0.04	0.353	81.84
Positive Control	120 uM		0.387	0.521	0.406	0.357	0.395		0.413	0.06	0.392	90.97
Negative Control	5000 uM		0.365	0.384	0.419	0.444	0.339	0.414	0.394	0.04	0.373	86.56
Test Item	C1	66.7 uM	0.460	0.345	0.328				0.378	0.07	0.357	82.73
	C2	80.1 uM	0.382	0.386	0.353				0.374	0.02	0.353	81.80
	C3	96.1 uM	0.439	0.326	0.437				0.401	0.06	0.380	88.06
	C4	115.0 uM	0.483	0.459	0.354				0.432	0.07	0.411	95.33
	C5	138.0 uM	0.021	0.017	0.017				0.018	0.00	-0.003	0.00*
	C6	166.0 uM	0.017	0.017	0.017				0.017	0.00	-0.004	0.00*

 

 

Table 5: Results of the main experiment 2 (fold induction)

           

Treatment Group	Concentration		Luminescence						Mean Luminescence	SD Luminescence	Mean Luminescence blank corr.	Fold Induction
			Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank			192
Solvent control									281.5	20.15	89.5	1.00
Medium control									288.5	18.74	96.5	1.08
Positive Control	120 uM		576	613	606	658	606		611.8	29.52	419.8	4.69
Negative Control	5000 uM		266	259	259	266	229	251	255.0	13.90	63.0	0.70
Test Item	C1	66.7 uM	443	288	266				332.3	96.47	140.3	1.57
	C2	80.1 uM	362	266	273				300.3	53.52	108.3	1.21
	C3	96.1 uM	347	273	266				295.3	44.88	103.3	1.16
	C4	115.0 uM	310	288	259				285.7	25.58	93.7	1.05
	C5	138.0 uM	259	266	214				246.3	28.22	54.3	0.61
	C6	166.0 uM	222	244	222				229.3	12.70	37.3	0.42

The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was 7.2% in the main experiment 2

Interpretation of results:

other: no activation of keratinocytes

Conclusions:

In conclusion, the test item did not activate the LuSens cells up to a concentration of 166 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP) of the test item.

In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 uM up to the highest tested concentration of 2000 uM (threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 138.1 uM.

 

The test item was tested in 2 independent main experiments.

 

The following concentrations of the test item were tested in the main experiments:

 

66.7, 80.1, 96.1, 115, 138, 166 |uM.

 

After treatment with the test item for 48 ± 1 hours the luciferase induction was above (>) 1.5 fold compared to the solvent control in 2 consecutive non-cytotoxic tested concentrations in the main experiment 1. A T-Test was performed for the statistical evaluation of the two consecutive non-cytotoxic tested concentrations with a luciferase induction of > 1.5 fold. The average luciferase activity induction were not statistically significant.

In the main experiment 2 the luciferase induction was not above or equal to 1.5 fold compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations, which confirms the negative result of the main experiment 1. Therefore, the LuSens prediction is considered negative.

 

The acceptance criteria were met:

• The average luciferase activity induction obtained with the positive control, 120 uM EGDMA was > 2.5 (ME 1: 5.05; ME 2: 4.69).


• The positive control had a relative cell viability > 70% as compared to the solvent control (ME 1: 70.17%; ME 2: 90.97%).


• The average luciferase activity induction obtained with the negative control, 5000 uM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.14; ME 2: 0.70).


• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 5.4%; ME 2: 7.2%).

At least three test concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability < 70%.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-07

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:

2020-06-26

Deviations:

yes

Remarks:

pretest to OECD 442C (solubility)

Principles of method if other than guideline:

pretest to OECD 442C

GLP compliance:

no

Remarks:

pretest to OECD 442C (solubility)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

SOLUBILITY TEST
- Solvent: Acetonitrile
- Test item preparation: 100 mM of the test item was prepared in acetonitrile.

Test 1: 100 μL of the 100 mM solution were added to 1500 μl buffer pH 7.5 and 400 μL acetonitrile and incubated for 24 h at room temperature.

Test 2: 500 μL of the 100 mM solution were added to 1500 μl buffer pH 10.2 and incubated for 24 h at room temperature.

Key result

Run / experiment:

other: Test 2

Parameter:

other: turbidity

Remarks on result:

other: turbid, after 24 h at RT: turbid

Key result

Run / experiment:

other: Test 1

Parameter:

other: turbidity

Remarks on result:

other: slightly turbid, after 24 h at RT: slightly turbid

Interpretation of results:

other: not applicable

Conclusions:

The test item is considered not suitable for the DPRA due to the poor water solubility of the test item under the conditions of this study.

Executive summary:

This study was performed to assess the solubility of the test item to conduct a DPRA. An appropriate solvent should dissolve the test item completely. To test the solubility, a test item concentration of 100 mM was clearly resolved in acetonitrile. After addition to the buffers pH 7.5 and pH 10.2, turbid liquids were formed due to the poor water solubility of the test item. During a 24-hour incubation at room temperature, the test item settled to the bottom. After strong shaking, both formulations appeared turbid again.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DPRA, WoE

This study was performed to assess the solubility of the test item to conduct a DPRA. An appropriate solvent should dissolve the test item completely. To test the solubility, a test item concentration of 100 mM was clearly resolved in acetonitrile. After addition to the buffers pH 7.5 and pH 10.2, turbid liquids were formed due to the poor water solubility of the test item. During a 24-hour incubation at room temperature, the test item settled to the bottom. After strong shaking, both formulations appeared turbid again. Thus, this study was stopped after pre-experiments.

 

ARE-Nrf2 Luciferase Test, WoE

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP) of the test item. In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 uM up to the highest tested concentration of 2000 uM (threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 138.1 uM. The test item was tested in 2 independent main experiments. The following concentrations of the test item were tested in the main experiments: 66.7, 80.1, 96.1, 115, 138, 166 |uM.

After treatment with the test item for 48 ± 1 hours the luciferase induction was above (>) 1.5 fold compared to the solvent control in 2 consecutive non-cytotoxic tested concentrations in the main experiment 1. A T-Test was performed for the statistical evaluation of the two consecutive non-cytotoxic tested concentrations with a luciferase induction of > 1.5 fold. The average luciferase activity induction were not statistically significant. In the main experiment 2 the luciferase induction was not above or equal to 1.5 fold compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations, which confirms the negative result of the main experiment 1. Therefore, the LuSens prediction is considered negative.

 

QSAR, WoE

The skin sensitising properties were estimated using Derek Nexus v. 6.0.1. No skin sensitising properties were estimated based on the described QSAR method (Derek, 2020).

The adequacy of a prediction depends on the following conditions:

1. a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;


2. b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;


3. c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;


4. d) the (Q)SAR model is relevant for the regulatory purpose.

 

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

 

Conclusion: Based on the ARE-Nrf2 Luciferase Test and the QSAR prediction, the test item is not considered to possess skin sensitising potential.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, it is concluded that the test item does not have skin sensitising potential and therefore shall not be classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.