Cyclopentene

Endpoint summary

Description of key information

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item Cyclopentene. Additionally, the query structure does not contain any unclassified or misclassified features and cyclopentene is consequently predicted to be a non-sensitizer.

A valid DPRA test was performed according to OECD TG 442C and GLP principles. For the DPRA assay Cyclopentene was dissolved in acetonitrile at 100 mM. Upon preparation of the SPCC and SPCL test item samples, a phase separation was observed which was no longer present in any of the samples after incubation.No co-elution of the test item with SPCC or SPCL 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 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.3% 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.

A valid KeratinoSens^TM assay was performed according to OECD TG 442D and GLP principles. For the KeratinoSens^TM assay Cyclopentene was dissolved in DMSO to a final concentration of 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. The test item only precipitated at the highest dose level tested in experiment 1. Two independent experiments were performed, which both passed the acceptance criteria. Cyclopentene showed no toxicity (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.16-fold and 1.22-fold in experiment 1 and 2 respectively. Cyclopentene is classified as negative in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

DEREK NEXUS – skin sensitization

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

Date of QSAR Prediction Reporting Format (QPRF): 21 March 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE : DEREK NEXUS
Date of QPRF: 21 March 2018
QPRF author and contact details: Charles River Laboratories Den Bosch

2. MODEL (incl. version number) : version 6.0.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL :
QSAR Prediction Reporting Format (QPRF):
- CAS number: 142-29-0
- EC number: 205-532-9
- Chemical name: Cyclopentene
- Structural formula: C5H8
- Structure codes:
- SMILES: C1C=CCC1
- InChI: Not available
- Other structural representation: mol file used:

Stereochemical features: DEREK prediction does take stereochemistry into account, if evidence of its influence on reactivity has been presented in literature; otherwise only 2D connectivity is taken into consideration. In this molecule no stereochemistry is present.

If a substance is predicted to be no skin sensitizer, DEREK NEXUS contains an expert-derived functionality to provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitization alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of Ames test or skin sensitization data, producing the following outcomes:
• In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.
• For those query compounds where features in the molecule are found in non-alerting skin sensitizers in the Lhasa reference set, the prediction remains negative and the misclassified[1]features are highlighted to enable the negative prediction to be verified by expert assessment.
•In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified[2]features are highlighted to enable the negative prediction to be verified by expert assessment.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Endpoint (OECD Principle 1)
Endpoint: Skin sensitization.
Dependent variable: Data from several toxicity assays (e.g. LLNA, GPMT, HRIPT) and mechanistic studies (e.g. DPRA) are synthesised to arrive at an expert conclusion of whether compounds within the model training set are likely to be a skin sensitizer.

Algorithm (OECD Principle 2)
Model or submodel name: DEREK NEXUS – skin sensitization.
Model version: DEREK NEXUS 6.0.1.
Reference to QMRF: The QMRF DEREK NEXUS 6.0 – skin sensitization was prepared by the provider LHASA Ltd (UK).
Predicted value (model result): Assessment with DEREK NEXUS did not yield any skin sensitization alerts for this structure.
Predicted value (comments): Not relevant; the structure did not fall within one of the currently 90 skin sensitization alerts, the structure contains no misclassified or unclassified features and consequently the structure is predicted to be negative.
Descriptor values: Not relevant.

Applicability domain (OECD principle 3)
Domains:
descriptor domain: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may
be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
structural fragment domain: for skin sensitization, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, “no alerts fired” may be extrapolated to a negative prediction. The structure contains no misclassified or unclassified features and consequently the structure is predicted to be negative.
mechanism domain: as the prediction is “no alerts fired” none of the mechanisms for skin sensitization is predicted to be applicable to this structure.
metabolic domain: not applicable
Structural analogues: Not applicable
Considerations on structural analogues: Not applicable

The uncertainty of the prediction (OECD principle 4)
DEREK NEXUS predictive performance against a combined human dataset had an accuracy of 76%. plesee refer to the link: https://www.lhasalimited.org/Public/Library/2014/Derek%20Nexus%20predicts%20human%20skin%20sensitisation%20accurately.pdf

The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5).
Not relevant, DEREK NEXUS did not yield any alerts for skin sensitization.

Adequacy (Optional)
Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments.
Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.

Qualifier:

no guideline required

Principles of method if other than guideline:

DEREK NEXUS – valid (Q)SAR model for skin sensitization

GLP compliance:

no

Remarks:

QSAR

Justification for non-LLNA method:

DEREK NEXUS – valid (Q)SAR model for skin sensitization

Specific details on test material used for the study:

Chemical name: Cyclopentene
CAS Number: 142-29-0
EC Number: 205-532-9
Molecular weight: 68.117 g/mol
Molecular formula: C5H8

The result as generated by DEREK NEXUS is presented in Appendix 2. The relevant QSAR Model Reporting Format (QMRF) is presented in the attached background material, as well.

Interpretation of results:

other: negative prediction for skin sensitisation

Conclusions:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item, Cyclopentene . Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. Cyclopentene is predicted to be not sensitizing to the skin. Substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH. The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitization.

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used. DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures. The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.  

If a substance is predicted to be a skin sensitizer, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:

EC3Q =  MWQ  /(Σ ωNN / Σ TNN)

MW = molecular weight

T = Tanimoto similarity score

ω = weighting factor = (MWNN/EC3) * TNN

Q = query compound

NN = nearest neighbour

The EC3 is the estimated concentration needed to produce a stimulation index (SI) of 3.

 

Conclusion:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item,Cyclopentene. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. Cyclopentene is predicted to be not sensitizing to the skin.

No EC3 value has been derived.

Cyclopentene should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH. The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitization.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2018 - 24 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours of incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at
220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Of the three human skin sensitisation tests that reflect Key Events in the Adverse Outcome Pathway (AOP), the Direct Peptide Reactivity Assay (DPRA) assay represents Key Event 1, Covalent binding of the electrophilic substance to proteins. Test materials are classified based on the results of “two out of three” tests.
The the DPRA assay is fully accepted at a regulatory level for the hazard identification of Skin Sensitisers and Non-Sensitisers in accordance with the UN GHS. This is fully approved by the EU Cosmetics Regulation 1223/2009, CLP Regulation 1272/2008 and REACH (Registration, Evaluation, Authorisation and restriction of CHemicals) legislation.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Appearance: Clear colourless liquid
- Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
- Batch No.of test material: 0000015386
- Expiration date of the batch: 19 March 2021
- Purity: 99.28%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In redrigerator (2-8°C)
- Stability under storage conditions: Yes until 30 November 2021

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Solubility of the test material in an appropriate solvent was assessed before the DPRA was performed. Acetonitrile (ACN), Milli-Q water (MQ); ACN:MQ (1:1, v/v); isopropanol, acetone: ACN (1:1 v/v) and dimethylsulphoxide (DMSO):ACN (1:9, v/v) were evaluated.
- Test material stock solutions were prepared freshly for each reactivity assay.
- 13.30 mg test material was pre-weighed into a clean amber glass vial and dissolved in 1953 μL ACN prior to use to obtain a 100 mM solution. Visual inspection of a clear soltion was considered sufficient to confirm that the test material was dissolved.
- Preliminary purification step (if any): No correction for the purity or composition of the test item.
- Final dilution of a dissolved solid, stock liquid or gel: 100 mM solution (cyclopentene in ACN)
- Samples of the test material, positive controls and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay. Any residual volumes were discarded.

Details on the study design:

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

TEST SYSTEM
- Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight for SPCC was 750.9 g/mol and for SPCL it was 775.9 g/mol.

CYSTEINE REACTIVITY ASSAY
> Preparation of solutions: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/ml) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixutre was stirred for 5 minutes and then sonicated for 5 more minutes.
> SPCC Reference Control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcys B, RCcysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCC stock solution with 250 μL ACN.
> SPCC Calibration curve: please refer to Table 1.
> The co-elution (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared as seen in Table 2.

LYSINE REACTIVITY ASSAY
> Preparation of solutions: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 then stirred for 5 minutes.
> SPCL Reference Control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB, RClysC) were prepared in amber vials by mixing 750 μL of the 0.667 mM SPCL stock solution with 250 μL ACN.
> SPCL Calibration Curve: please refer to Table 3.
> The co-elution (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared as seen in Table 4.

SAMPLE INCUBATIONS
- After preparation, all samples (including reference, control and calibration samples) were placed in the autosampler in the dark and incubated at 25±2.5°C.
- The incubation time from the placing the samples in the autosampler and the analysis of the first RCcysB- or RClysB-sample was 25 hours.
- The time between the first RCcysB- or RClysB-injectin and the last injection of a cysteine or lysine sequence did not exceed 30 hours.
- Samples were visually inspected for precipitation before HPLC-PDA analysis.

HPLC-PDA Analysis
- SPCC and SPCL peak areas in the samples were measured by HPLC-PDA.
- Sample analysis was performed according to the the systems show in Figure 1.

Positive control results:

CYSTEINE ASSAY: The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 68.2% ± 0.5%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

LYSINE ASSAY: The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was57.3% ± 3.4%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Key result

Run / experiment:

other: Mean ± SD

Parameter:

other: SPCC Depletion

Value:

3.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ± SD 1.7%

Remarks:

Negative DPRA Prediction: No or minimal reactivity

Key result

Run / experiment:

other: Mean ± SD

Parameter:

other: SPCL Depletion

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ± SD 2.5%

Remarks:

Negative DPRA Prediction: No or minimal reactivity

Key result

Run / experiment:

mean

Parameter:

other: Mean of SPCC and SPCL depletion

Value:

2.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative DPRA depletion

Remarks:

No or minimal reactivity

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
- Range of historical values if different from the ones specified in the test guideline: Yes

SOLUBILITY ASSESSMENT OF TEST MATERIAL

- At a concentration of 100 mM, cyclopentene was not soluble in MQ, ACN:MQ (1:1 v/v), acetone:ACN (1:1, v/v) and DMSO:ACN (1:9, v/v), but was soluble in ACN and isopropanol.

- ACN is the preferred solvent for DPRA and so was used to dissolve the test material in this study.

ACCEPTABILITY OF CYSTEINE REACTIVITY ASSAY

- The correlation coefficient (r-squared) of the SPCC standard calibration curve was 0.997. The standard calibration curve was accepted since the r-squared value was > 0.99.

- The mean peptide concentrations of Reference Controls A (0.510 ±0.07 mM) and Reference Controls C (0.499 ±0.007 mM) were both within the acceptance range of (0.50 ±0.05 mM), confirming the suitability of the HPLC system and indicating that the ACN solvent did not affect the Percent SPCC Depletion.

- The Coefficient of Variation (CV) of the peptide areas for the 9 Reference Controls B and C was 2.3%, which was within the acceptance criteria (CV < 15%) and thus confirms the stability of the HPLC run over time.

- The mean area ratio (A220/A258) of the Reference Control samples was 18.12. The mean A220/A258 ratio ± 10% range was 16.31-19.94. Each sample showed an A220/A258 ratio within this range, signifying that co-elution has not occurred.

> RESULTS

- Preparation of a 100mM cyclopentene stock solution in ACN showed that the test material dissolved completely.

- Upon preparation, a phase separation was observed in the co-elution control (CC) and test material samples, but was not seen after incubation of the test material samples.

- In the CC sample, there was no peak observed at the retention time of SPCC, which showed that there was no co-elution of the test material with SPCC.

- For the 209393/A-cys samples, the mean SPCC A220/A258 area ratio was 18.35, which was within the 16.31-19.94 range, further corroborating that there was no co-elution.

- The mean Percent SPCC Depletion for the test item was 3.1% ± 1.7%.

ACCEPTABILITY OF CYSTEINE REACTIVITY ASSAY

- The correlation coefficient (r-squared) of the SPC; standard calibration curve was 0.991. The standard calibration curve was accepted since the r-squared value was > 0.99.

- The mean peptide concentrations of Reference Controls A (0.510 ±0.020 mM) and Reference Controls c (0.520 ±0.003 mM) were both within the acceptance range of (0.50 ±0.05 mM), confirming the suitability of the HPLC system and indicating that the ACN solvent did not affect the Percent SPCC Depletion.

- The Coefficient of Variation (CV) of the peptide areas for the 9 Reference Controls B and C was 3.3%, which was within the acceptance criteria (CV < 15%) and thus confirms the stability of the HPLC run over time.

- The mean area ratio (A220/A258) of the Reference Control samples was 16.14. The mean A220/A258 ratio ± 10% range was 14.52-17.75. Each sample showed an A220/A258 ratio within this range, signifying that co-elution has not occurred

> RESULTS

- Preparation of a 100mM cyclopentene stock solution in ACN showed that the test material dissolved completely.

- Upon preparation, a phase separation was observed in the co-elution control (CC) and test material samples, but was not seen after incubation of the test material samples.

- In the CC sample, there was no peak observed at the retention time of SPCC, which showed that there was no co-elution of the test material with SPCC.

- For the 209393/A-cys samples, the mean SPCC A220/A258 area ratio was 16.32, which was within the 14.52 -17.75 range, further corroborating that there was no co-elution.

- The mean Percent SPCC Depletion for the test item was 1.4% ± 2.5%.

Table. 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		Cysteine 1:10 / Lysine 1:50 prediction model
Cyclopentene	3.1%	±1.7%	1.4%	±2.5%	2.3%	Negative: No or minimal reactivity

SD = Standard Deviation

Interpretation of results:

GHS criteria not met

Conclusions:

Cyclopentene was found to be negative in the DPRA assay. In the cysteine reactivity assay the test material showed 3.1% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.3% and as a result the test item 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.

Executive summary:

The aim of this study was to determine the reactivity of the test material, Cyclopentene, towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL), according OECD 442C guideline.

The test material was dissolved in acetonitrile to obtain a final 100 mM solution. The solubility of the test material in several solvents was investigated prior to conducting the DPRA assay. Following incubation of the test material with either SPCC or SPCL, the relative peptide cocnentration was determined by HPLC with gradient elution and photodiode array (PDa) detection at 220 nm and 258nm. SPCC and SPCL percent depletion values were calculated and used in the Cysteine 1:10/Lysine 1:50 prediction model, which allows the test material to be assigned to one of four reactivity classes in order to be able to discriminate between sensitisers and non-sensitisers.

The validation parameters were all within the acceptability criteria for the DPRA. A phase separation was observed in the SPCC and SPCL test material samples upon preparation, but disappeared following incubation.

In conclusion, this DPRA test was considered to be valid. Cyclopentene was found to be negative in the DPRA assay. In the cysteine reactivity assay the test material showed 3.1% SPCC depletion while in the lysine reactivity assay the test item showed 1.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.3% and as a result the test item 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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 May 2018 to 31 May 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)

Deviations:

yes

Remarks:

Based on laboratory historical data, the deviations are considered not to affect the study integrity.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Of the three human skin sensitisation tests that reflect Key Events in the Adverse Outcome Pathway (AOP), the KeratinoSens™ test represents Key Event 2, the activation of skin cells (keratinocytes) in the epidermis. Test materials are classified based on the results of “two out of three” tests. The the KeratinoSens™ test is fully accepted at a regulatory level for the hazard identification of Skin Sensitisers and Non-Sensitisers in accordance with the UN GHS. This is fully approved by the EU Cosmetics Regulation 1223/2009, CLP Regulation 1272/2008 and REACH (Registration, Evaluation, Authorisation and restriction of CHemicals) legislation.

Specific details on test material used for the study:

TEST MATERIAL INFORMATION
Chemical name (IUPAC, synonym or trade name): Cyclopentene
CAS number: 142-29-0
EC Number: 205-532-9
Molecular formula: C5H8
Molecular weight: 68.117
Highly flammable: Yes
Solubility in vehicle: Dimethyl sulfoxide: Not indicated
Stability in vehicle: Dimethyl sulfoxide: Not indicated
Identification: Cyclopentene
Appearance: Clear colourless liquid
Batch: 0000015386
Purity/Composition: 98.9% (Information extracted from the Certificate of Analysis)
Test item storage: In refrigerator (2-8°C)
Stable under storage conditions until: 19 March 2021 (expiry date)
Test Facility test item number: 209393/A
Purity/Composition correction factor: No correction factor required
Test item handling: The test item is volatile and evaporates rapidly at room temperature. Do not leave the test item at room temperature longer than 30 minutes.
Stability at higher temperatures: Stable at temperatures << 44°C. Not stable at higher temperatures, potential to polymerize and form peroxides

Details on the study design:

DOSE FORMULATION
The test item, stored in the fridge, was taken out, weighed within 5 min and formulated within 10 min in experiment 1. It was then weighed within 3 min and formulated within 56 min in experiment 2.
A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless).
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 – as recommended in the OECD 442D guideline).

In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) several times 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. Precipitation was only observed at the end of the incubation period in the 96-well plates at the highest test concentration in experiment 1. Test item concentrations were used within 2.5 hours after preparation.

PREPARATION OF THE POSITIVE CONTROL
Positive control: Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands). The final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

PREPARATION OF THE VEHICLE CONTROL
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

BLANK
On each plate three blank wells were tested (no cells and no treatment).

TEST SYSTEM:
The test system used is 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) (Obtained from Givaudan (Duebendorf, Switserland). 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 from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

MAINTANCE MEDIUM
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).

EXPOSURE MEDIUM
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

ENVIRONMNTAL CONDITIONS
Humid atmosphere: 80 - 100% (actual range 72 – 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 – 37.2 °C).
Temperature and humidity: continuously monitored throughout the experiment.
CO2 percentage: monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical
data these deviations are considered not to affect the study integrity.

SUBCULTURING
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 (P+25).

EXPERIMENTAL DESIGN
PLATING
Test: 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 for approximately 24 hours in the incubator. The passage number used was P+8 in experiment 1 and P+13 in experiment 2.

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±1.0
O C in the presence of 5% CO2.

LUCIFERASE ACTIVITY MEASURAMENT
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) 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 ASSAY
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; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 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 (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm.

Positive control results:

Both Experiment 1 and 2 met the acceptance criteria:

1. The Ethylene dimethacrylate glycol value was statistically significant above the threshold of 1.5-fold in at least one concentration.

2. The EC1.5 of the positive control was between 5 and 125 µM (33 µM and 46 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (5.76-fold and 3.43-fold in experiment 1 and 2, respectively).

In Experiment 1, the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 5.76 and the EC1.5 33 µM.

In Experiment 2, The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.43 and the EC1.5 46 µM.

Key result

Run / experiment:

other: 1

Parameter:

other: viability of the cells (%)

Value:

70

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

No activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway.

Key result

Run / experiment:

other: 2

Parameter:

other: viability of the cells (%)

Value:

70

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

No activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway.

Other effects / acceptance of results:

OTHER EFFECTS:
- No precipitation was observed in both Experiment 1 and Experiment 2 at the start of the incubation period in the 96-well plates. At the end of the incubation period precipitation was observed only at the highest test concentration in the 96-well plates.

ACCEPTANCE OF RESULTS:
Both experiments passed the acceptance criteria:
**The luciferase activity induction obtained with 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 (33 µM and 46 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (5.76-fold and 3.43-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% (8.4% and 6.1% in experiment 1 and 2, respectively).

Attached in the "backgroud material" box are:

- Luminescence and Viability of the positive control in Exp 1 and Exp 2

- Luminescence and Viability of the test material in Exp 1 and Exp 2

- Overview Luminescence Induction abd Cell Viability of Cyclopentene in Exp 1 and 2

- Overview Luminescence Induction abd Cell Viability of the positive control EDMG in Exp 1 and 2

- Overview of EC 1.5, Imax, IC30 and IC50 Values

Interpretation of results:

GHS criteria not met

Conclusions:

The test material Cyclopentene CAS 142-29-0 / EC 205-532-9 showed no toxicity and no biologically relevant induction of the luciferase activity was measured at any of the test concentrations in both experiments. Therefore, Cyclopentene is classified as negative according to the results of this study.

Executive summary:

The aim of this study was to assess the ability of the test material Cyclopentene (CAS 142-29-0/EC 205-532-9) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay. The study was performed according to the OECD 442D guideline and under GLP conditions.

Skin sensitisation is defined as a series of key events, that form an adverse outcome pathway (AOP). The test methods here addressed is the second key event of the skin sensitisation AOP.

The test material was dissolved in dimethyl sulfoxide (DMSO) and the final concentrations were 0.98 – 2000 µM (2-fold dilution series). Two separated experiments were performed.

In the Experiment 1, precipitation was observed only at the end of the incubation period at the highest test concentration in the 96-well plates. The test material Cyclopentene showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated. No luminescence activity induction was observed at any of the test concentrations after treatment with Cyclopentene, compared to the vehicle control. The Imax was 1.16 and therefore no EC_1.5 could be calculated.

A dose related induction of the luciferase activity of the positive control Ethylene dimethacrylate glycol was observed and the Imax was 5.76 and the EC_1.5 33 µM. 

 

In the Experiment 2, no precipitation was observed at the start and end of the incubation period in the 96-well plates. Alos in this case, the test material Cyclopentene showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Cyclopentene. The Imax was 1.22 and therefore no EC_1.5 could be calculated. A dose related induction of the luciferase activity was observed with the positive control Ethylene dimethacrylate glycol. The I_max was 3.43 and the EC_1.5 46 µM. 

 

The acceptance criteria were met in both Experiment 1 and 2.

1. The luciferase activity induction of the positive control Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. 

2. The EC_1.5 of the positive control was between 5 and 125 µM (33 µM and 46 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (5.76-fold and 3.43-fold in experiment 1 and 2, respectively).

3. The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.4% and 6.1% in experiment 1 and 2, respectively).

 

In conclusion, the test material Cyclopentene (CAS 142-29-0/EC 205-532-9) is classified as negative in the KeratinoSens TM assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

As the substance is a mono-constituent and not a metal the following strategy was followed:

STEP 0: The starting point for the weight of evidence is the assessment whether new studies are required. Available information on the test item was used to evaluate whether:

·      The substance is not a strong acid (pH≤ 2.0) or base (pH ≥ 11.5), known to be not corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature.

·      No adequate existing human data, which provide evidence that the substance is a skin sensitizer are available.

·      No data from existing studies on skin sensitization in laboratory animals, which provide sound conclusive evidence that the substance is a sensitizer or non-sensitizer are available.

This step is the responsibility of the sponsor.

If no reliable data on solubility is available, a solubility test is performed at Charles River to determine whether the substance dissolves sufficiently in a solvent which is appropriate for each test mentioned below. In case the solubility test demonstrates solubility of the test substance that meets the precondition limits for thein vitrotests, the following step-wise testing approach is followed:

STEP 1: DEREK assessment (overall skin sensitizing events), Direct Peptide Reactivity Assay (DPRA; molecular interaction with skin proteins) and KeratinoSens^TM assay (inflammatory response in keratinocytes) are performed.

STEP 2: Depending on the outcome of the studies performed in STEP 1 and in absence of a definite conclusion on possible skin sensitization, the U-SENS^TM assay is performed (key event:activation of dendritic cells).

STEP 3: Based on a weight of evidence of all available data on the test item related to skin sensitization, an argument is prepared to conclude on the classification for the substance or, if no conclusion can be drawn, to conclude on the performance of anin vivoskin sensitization study.

No data were available that would preclude performance of the studies to determine the potential for skin sensitization for cyclopentene (CAS 142-29-0 / EC 205-532-9). Therefore, STEP 1 studies were performed, i.e. DEREK assessment (project no 20149486); DPRA assay (project no 20149488) and KeratinoSens^TMassay (project no 20149489).

Based on the DEREK NEXUS assessment in which Cyclopentene is predicted to be not sensitizing to the skin, a negative DPRA assay and a negative KeratinoSens^TM assay, Cyclopentene (CAS 142-29-0 / EC 205-532-9) can be concluded to have no skin sensitizing properties. Performance of a U-SENS^TM assay (STEP 2) is considered to give no additional information, the substance would be regarded as non-sensitizing irrespective of whether the result would be negative or positive (at least two out of three tests are negative).

Justification for classification or non-classification

Based on the DEREK NEXUS assessment in which Cyclopentene is predicted to be not sensitizing to the skin, a negative DPRA assay and a negative KeratinoSens^TM assay, Cyclopentene (CAS 142-29-0 / EC 205-532-9) can be concluded to have no skin sensitizing properties. Performance of a U-SENS^TM assay (STEP 2) is considered to give no additional information, the substance would be regarded as non-sensitizing irrespective of whether the result would be negative or positive (at least two out of three tests are negative).

In conclusion, Cyclopentene (CAS 142-29-0 / EC 205-532-9) does not have any skin sensitizing properties. The substance does not have to be classified according to Regulation 1272/2008 and amendments.