Reaction mass of m,alpha-dimethylstyrene and p,alpha-dimethylstyrene

Endpoint summary

Description of key information

A DEREK assessment, a DPRA assay and a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. The testing was done according to the most recent OECD guidelines and under GLP principles.

None of the tests indicated that Reaction mass of m, alpha-dimethylstyrene and p, alpha-dimethylstyrene has skin sensitising properties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

In silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

May 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

This study is part of a weight of evidence approach following ECHA guidance: Guidance on information requirements and chemical safety assessment Chapter R.7a Endpoint specific guidance v.6.0 July 2017, paragraph 7.3.

Qualifier:

no guideline followed

Principles of method if other than guideline:

DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitisation alerts.

GLP compliance:

no

Positive control results:

n.a.

Key result

Parameter:

other: structural alerts for skin sensitisation

Remarks on result:

no indication of skin sensitisation

The full report including the result as generated by DEREK NEXUS, the relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) is attached.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser. Reaction mass of m, alphadimethylstyrene and p, alpha-dimethylstyrene is predicted to be not sensitising to the skin.

Interpretation of results:

study cannot be used for classification

Remarks:

Study is part of a weight of evidence approach and is not used for classification on its own.

Conclusions:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for Reaction mass of m, alphadimethylstyrene and p, alpha-dimethylstyrene. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 July 2018 - 12 July 2018

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:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

No purity correction factor was required.

Details on the study design:

TEST ITEM PREPERATION
No correction for the purity/composition of the test item was performed.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. The test item should dissolve completely in an appropriate solvent, i.e. by visual inspection the solution has to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol (MeOH) and ethanol (EtOH).
Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 25.65 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1940 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM: Synthetic peptides containing cysteine (SPCC) (Ac- RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 23.5 hours and 23 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC-PDA analysis the samples were visually inspected for precipitation. The samples that showed a phase separation were centrifuged (at 400 g) for 5 minutes at room temperature.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials'). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

POSITIVE CONTROL: Cinnamic aldehyde
- Purity: 99.1%

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration, and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]*100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample a ratio in the range of 90%< mean area ratio of control samples <110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION (see also 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitiser and a non-sensitiser.

Positive control results:

The positive control had a mean SPCC depletion of 70.8% ± 2.2% and a mean SPCL depletion of 54.7% ± 2.9%.

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: SPCC mean depletion (%)

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: SD: 0.2%; Upon preparation a precipitate was observed in the CC and test item samples while after incubation a phase separation was observed.

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL mean depletion (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: SD: 0.0%; Upon preparation a precipitate was observed in the CC and test item samples while after incubation a phase separation was observed.

Other effects / acceptance of results:

Solubility of the test item:
- The test item showed to be soluble in ACN, isopropanol, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v), ethanol and methanol. Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this study. In the SPCC assay, solubility of the test item was assessed in the above mentioned solvents by mixing 50 μL of the 100 mM test item solution with 750 μL phosphate buffer pH 7.5 and 200 μL ACN followed by vortex mixing. For each of the selected solvents, formation of a precipitate was observed. In the SPCL assay, solubility of the test item was assessed in the above mentioned solvents by mixing 250 μL of the 100 mM test item solution with ammonium acetate buffer pH 10.2 followed by vortex mixing. For each of the selected solvents, formation of a precipitate was observed.

Co-elution and precipitation:
In both the SPCC and the SPCL assay, the co-elution control (CC) as well as the test item samples were visually inspected upon preparation and after incubation. Upon preparation a precipitate was observed in the CC and test item samples while after incubation a phase separation was observed. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

For details on results see tables included in 'Any other information on results incl. tables'.

SPCC and SPCL depletion and reactivity classification for the test item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	Reactivity class
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
m, alpha-dimethylstyrene and p, alpha-dimethylstyrene	0.3%	±0.2%	0.0%	±0.0%	0.1%	Negative: no or minimal reactivity

Acceptability of the DPRA assay

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPLC
Correlation coefficient (r^2) standard calibration curve	>0.99	0.995	>0.99	0.995
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.505 ± 0.002	0.50 ± 0.05	0.523±0.020
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.506 ± 0.001	0.50 ± 0.05	0.516±0.005
CV (%) for RC samples B and C	<15.0	0.5	<15.0	4.1
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	70.8	40.2-69.0	54.7
SD of peptide depletion cinnamic aldehyde (%)	<14.9	2.2	<11.6	2.9
SD of peptide depletion for the test item (%)	<14.9	0.2	<11.6	0.0

Interpretation of results:

other: Study is part of a weight of evidence approach and is not used for classification on its own.

Conclusions:

Reaction mass of m, alpha-dimethylstyrene and p, alpha-dimethylstyrene was negative in the DPRA, performed according to OECD 442C and GLP principles, and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since a phase separation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 June 2018 - 13 July 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)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitisation tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD). The purpose of this study was to evaluate the ability of Reaction mass of m, alpha-dimethylstyrene and p, alpha-dimethylstyrene to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. Activation of this pathway can lead to skin sensitisation.

Specific details on test material used for the study:

- No purity correction factor was required.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Number of replicates: two independent experiments were performed, each concentration was tested in triplicate for the luciferase activity measurements, one parallel replicate was used for an MTT cell viability assay.

CONTROLS:
Positive control: ethylene dimethacrylate glycol, 7.8-250 µM, tested in triplicate, DMSO was used as a vehicle;
Negative control: DMSO (1% in exposure medium);
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values.

TEST SYSTEM:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. The cells were incubated overnight in the incubator. The passage number used was P+9 in experiment 1 and P+11 in experiment 2.

TEST ITEM PREPARATION:
A solubility test was performed. The test item was dissolved in DMSO to a concentration of 200 mM (clear colorless solutions). The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed a clear solution (2000 μM). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM (clear colorless solutions). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates, except in the first experiment at the end of the incubation period where precipitate was observed at concentrations of 250 μM and upwards.
Test item concentrations were used within 2.5 hours after preparation.

TEST CONCENTRATIONS:
- In both experiments: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM

MEDIA:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

TREATMENT OF CELLS:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 ± 1 hours in a humid atmosphere of 80 - 100% (actual range 65 – 94%) in the dark at 37.0 ± 1.0°C (actual range 35.6 – 37.2°C), in the presence of 5% ± 0.5% CO2.

LUCIFERASE ACTIVITY MEASUREMENT:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

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

Positive control results:

The EC1.5 of the positive control was between 5 and 125 μM (81 μM and 24 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.38-fold and 3.79-fold in experiment 1 and 2, respectively).

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.15

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

1.18

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

- In the first experiment, precipitation was only observed at the end of the incubation at concentrations of 250 μM and upwards. In the second experiment, no precipitation of the test item was observed at the start and at the end of the incubation period.
- In both experiments, the test item showed toxicity: the calculated IC30 were 170 and 636 μM for experiment 1 and 2, respectively and the calculated IC50 were 207 and 740 μM for experiment 1 and 2, respectively.
- For both experiments, no luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. Based on the Imax values, no EC1.5 could be calculated.

Both experiments passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (81 μM and 24 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.38-fold and 3.79-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.5% and 12% in experiment 1 and 2, respectively).

Interpretation of results:

other: Study is part of a weight of evidence approach and is not used for classification on its own.

Conclusions:

Based on the outcome of a KeratinoSens™ assay performed according to OECD guideline and GLP principles, Reaction mass of m, alpha-dimethylstyrene and p, alpha-dimethylstyrene is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for Reaction mass of m, alphadimethylstyrene and p, alpha-dimethylstyrene. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser. Furthermore, Reaction mass of m, alpha-dimethylstyrene and p, alpha-dimethylstyrene was negative in the DPRA, therefore it can be concluded that the substance does not interfere with protein moieties. Finally, Reaction mass of m, alpha-dimethylstyrene and p, alpha-dimethylstyrene was found not to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes according to the results of a KeratinoSensTM assay. Performance of a USENSTM assay (Key event 3) is considered to give no additional information, the substance would be regarded as non-sensitising irrespective of whether the result would be negative or positive (at least two out of three tests are negative).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Based on the current data-set it is concluded that there are no indications that Reaction mass of m, alpha-dimethylstyrene and p, alpha-dimethylstyrene has skin sensitising properties. The data are considered sufficient to conclude that the substance does not have to be classified for skin sensitising properties according to Regulation 1272/2008 and amendments.