Indene

Endpoint summary

Description of key information

A DEREK assessment, DPRA assay and 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 outcome of these studies did not indicate skin sensitising properties of Indene.

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:

17 Jul 2018 - 10 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The existing knowledge of the chemical and biological mechanisms associated with skin sensitisation has been summarized in the form of an Adverse Outcome Pathway (AOP). The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways. The KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay) is performed to address this second key event. This test is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in vitro test to be performed as part of the testing strategy for the skin sensitisation endpoint.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Feb 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

DOSE FORMULATION
- A correction factor according to correct for the purity (96.6%) was applied in this study.
- A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM. This concentration was selected as the highest concentration for the main assay and it is the highest dose required in the current guideline.

MAIN EXPERIMENT
- The test item was dissolved in dimethyl sulfoxide (DMSO) at 200mM. 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.
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- Test item concentrations were used within 2.5 hours after preparation.

CONTROL ITEMS
- Vehicle: dimethyl sulfoxide (DMSO)
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

- Positive control: Ethylene dimethacrylate glycol
Preparation of the positive control: 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO. The final concentration ranges from 7.8 to 250 μM (final concentration DMSO of 1%).
All concentrations of the positive control were tested in triplicate.

- Blank control: On each plate three blank wells were tested (no cells and no treatment).

TEST DESIGN
- Test system: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
- Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.
- 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 items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37 ± 1°C in the presence of 5% CO2. In total 2 valid experiments were performed.
- Luciferase activity measurement: 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 KeratinoSens™ 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 - 4 hours at 37°C, 5% CO2. The MTT medium was then removed and cells were lysed, subsequently the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

DATA ANALYSIS

Interpretation
The following parameters are calculated in the KeratinoSensTM test method:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control.
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained.
- The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Acceptance criteria
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 should be between 5 and 125 μM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.70 and the EC 1.5 was 55 μM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.35 and the EC 1.5 was 18 μM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.47

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: EC1.5: not possible to calculate

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.03

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: EC1.5: not possible to calculate

Other effects / acceptance of results:

ACCEPTANCE: both tests 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 (55 μM and 18 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.70-fold and 2.35-fold in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (18% and 13% in experiment 1 and 2, respectively).

CYTOTOXICITY: yes

Experiment 1:
IC30:671 μM
IC50: 813 μM.

Experiment 2:
IC30: 785 μM
IC50: 993 μM.

PRECIPITATION: no
No precipitation was observed at the start and end of the incubation period.

Interpretation of results:

other: Study cannot be used for classification independently, but in a WoE for the end point Skin Sensitisation.

Conclusions:

A KeratinoSens™assay was performed according to OECD 442D guideline and following GLP principles to assess the skin sensitisation hazard potential. Indene was classified as negative in the Keratinosens assay since no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes was observed.

Executive summary:

A KeratinoSens™assay was performed according to OECD 442D guideline and following GLP principles to assess the skin sensitisation hazard potential of Indene. Indene was dissolved in dimethyl sulfoxide and tested at 4-fold dilution series from 0.98 µM to 2000 µM. Two independent experiments were performed. Both experiments passed the acceptance criteria and the test conditions were adequate, implying that the test system functioned properly. Indene showed toxicity (IC30 values of 671μM and 785μM and IC50 values of 813μM and 993μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. In conclusion, Indene was found to be negative in the Keratinosens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000μM.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 Jul 2018 - 17 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The DPRA assay is a validated in chemico skin sensitisation test, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of a weight of evidence approach.

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)

Details on the study design:

TEST ITEM PREPARATION
A correction factor was made for purity/composition of the test item. A correction factor of (1/0.966=) 1.035 was used.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had 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) and dimethylsulfoxide (DMSO):ACN (1:9, v/v).
Test item stock solutions were prepared freshly for each reactivity assay.
For the cysteine and lysine reactivity assay respectively 12.89 mg and 22.10 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1072 μL and 1838 μL ACN, respectively, to obtain 100 mM solutions. 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)
Molecular weight: 750.9 g/mol
- Synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH)
Molecular weight: 775.9 g/mol.

Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Storage: The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.

POSITIVE CONTROL
Cinnamic aldehyde
Appearance: Yellow liquid
CAS Number: 104-55-2
Molecular Weight: 132.16 g/mol
Purity: 99.1%
Storage: At room temperature
Purity/composition correction factor: No correction factor required

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%
DATA INTERPRETATION
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 (see table below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Positive control results:

The mean Percent SPCL Depletion for the cinnamic aldehyde was 52.7% ± 4.0%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum allowed (SD <11.6%).

Key result

Parameter:

other: Mean SPCC depletion (%)

Value:

3.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(CV between reference controls: 1.0%)

Positive controls validity:

valid

Remarks:

(Mean percentage SPCC: 69.5% ± 1.0%)

Key result

Parameter:

other: Mean SPCL depletion (%)

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(CV between reference controls: 3.3%)

Positive controls validity:

valid

Remarks:

(Mean percentage SPCL: 52.7% ± 4.0%)

Other effects / acceptance of results:

Upon preparation of the SPCC test item samples no precipitation or phase separation was observed in any of the samples.
Upon preparation of the SPCL test item samples a precipitate was observed.
After incubation of the SPCC and SPCL test item samples, a phase separation was observed.

 SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD	1.7%	Cysteine 1:10 / Lysine 1:50 prediction model
Indene	3.1%	±0.7%	0.3%	±0.5%		Negative: No or minimal reactivity

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r2) standard calibration curve	>0.99	0.994	>0.99	0.9990
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.515 ± 0.010	0.50 ± 0.05	0.513 ± 0.022
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.515 ± 0.005	0.50 ± 0.05	0.512 ± 0.009
CV (%) for RC samples B and C	<15.0	1.0	<15.0	3.3
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	69.5	40.2-69.0	52.7
SD of peptide depletion cinnamic aldehyde (%)	<14.9	1.0	<11.6	4.0
SD of peptide depletion for the test item (%)	<14.9	0.7	<11.6	0.5

Interpretation of results:

other: Study cannot be used for classification independently, but in a WoE for the endpoint Skin Sensitisation

Conclusions:

An in chemico DPRA study was performed according OECD guideline 422C and following GLP principles. Indene was negative in the DPRA 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, it is not certain how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with caution.

Executive summary:

In an in chemico study performed according OECD guideline 422C and following GLP principles, the reactivity of Indene towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined. The validation parameters were all within the acceptability criteria for the DPRA, therefore the study was considered to be valid. Upon preparation of the SPCC test item samples no precipitation or phase separation was observed in any of the samples. Upon preparation of the SPCL test item samples a precipitate was observed. In the cysteine reactivity assay the test item showed 3.1% SPCC depletion while in the lysine reactivity assay the test item showed 0.3% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.7% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation and phase separation were observed after the incubation period for both SPCC and SPCL, this negative result is uncertain and should be interpreted with caution.

Reference 3

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 Indene. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DEREK NEXUS version 6.0.1 did not yield any alert for skin sensitisation for Indene. It is of note that the query structure contains a feature that was not found in the Lhasa skin sensitisation negative prediction dataset (unclassified). Furthermore, Indene was negative in the DPRA, therefore it can be concluded that the substance does not interact with protein moieties. Finally, Indene 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 U-SENSTM 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 study available

Justification for classification or non-classification

Based on the current data-set it is concluded that there are no indications that Indene 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.