4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone

Endpoint summary

Description of key information

The in vitro DPRA and KeratinoSens assay are both negative. These are in agreement with the old in vivo studies by Klecak where no sensitisation was observed.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

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))

Qualifier:

according to guideline

Guideline:

other: ECVAM (2014), DB-ALM protocol 154: Direct peptide reactivity assay (DPRA) for skin sensitization testing

Principles of method if other than guideline:

​Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 4 years experience with both the KeratinoSens and DPRA assays.

GLP compliance:

no

Remarks:

Conducted within the "spirit" of GLP (See Principles of method if other than guideline)

Type of study:

other: Direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Trivial name : KEPHALIS
Chemical name : Cyclohexanone, 4-(1-ethoxyethenyl)-3,3,5,5-tetramethyl-
Molecular weight : 224.3
Molecular formula : C14H24O2
Purity : 80.9 % (sum of two main peaks)
Supplier : Givaudan Schweiz AG
Product code : 6378003
CAS number : 36306-87-3
EC number : 252-961-2
Expiration date : 19 Oct 2016
Batch number : PE00141817
Physical form : Liquid
Storage conditions : 4°C

Details on the study design:

The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test a substance towards peptides.
This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

Experimental
The test substance KEPHALIS was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by KEPHALIS was determined by HPLC-UV.

Test System(s):
The Lys-peptide Ac-RFAAKAA is incubated at a final concentration of 0.5 mM in an ammonium acetate buffer at pH 10.5 in presence of a final level of 25% acetonitrile and in presence of a 50-fold excess of the test substance (25 mM) dissolved in acetonitrile.
The Cys-peptide Ac-RFAACAA is incubated at a final concentration of 0.5 mM in phosphate buffer at pH 7.5 in presence of a final level of 25% acetonitrile and in presence of a 10-fold excess of the test substance (5 mM) dissolved in acetonitrile.

Endpoint & Endpoint Detection:
24 h after start of the incubation the remaining peptide is quantified with HPLC-UV.

Endpoint Value:
The endpoint is expressed as % peptide depletion.

Positive control
In each test Cinnamic aldehyde is included as positive control.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP.

Prediction Model
Based on the average peptide depletion values for both the Cysteine and the Lysine peptide, a reactivity class is attributed to the substances. Average depletion below 6.38% indicates minimal reactivity, substances in this class are predicted as non-sensitizers by the DPRA.
The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is described in Test report RCR 153’453, ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the Givaudan testing facility’.

Parameter:

other: Cys-peptide depletion

Value:

2.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Lys-peptide depletion

Value:

4.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Average depletion Cys-and Lys-peptide

Value:

3.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Elution time Cys peptide

Value:

10.64

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Elution time test substance Cys peptide run

Value:

15.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Elution time Lys peptide

Value:

7.97

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Elution time test substance Lys peptide run

Value:

16.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test substance overall results:

	Average	Standard deviation
Cys-peptide depletion	2.9	2.6
Lys-peptide depletion	4.1	0.1
Average depletion Cys-and Lys-peptide	3.5
Reactivity Class	MINIMAL
Prediction	Non-sensitizer
Elution time Cys peptide	10.64
Elution time test substance Cys peptide run	15.5 / 16.1
Elution time Lys peptide	7.97
Elution time test substance Lys peptide run	16.1

Interpretation of results:

GHS criteria not met

Conclusions:

KEPHALIS was non-reactive and classified into the MINIMAL reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

Executive summary:

The result of the DPRA assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the KeratinoSens™ assay may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of chemicals, two congruent results in these two tests give a good prediction of the sensitizer hazard [3-5] particularly when predicting human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results. KEPHALIS was non-reactive and classified into the MINIMAL reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Qualifier:

according to guideline

Guideline:

other: 2014. EURL ECVAM Recommendation on the Keratinosens Assay for Skin Sensitisation Testing

Qualifier:

according to guideline

Guideline:

other: DB-ALM protocol 155: KeratinosensTM protocol

Principles of method if other than guideline:

The KeratinosensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their abiliyt to induce the Nrf2-response.
​Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 4 years experience with both the KeratinoSens and DPRA assays.
DEVIATION FROM STANDARD PROTOCOL
According to standard protocol, cytotoxicity is measured by MTT test in a parallel plate performed at the same time the luciferase assay is performed. In a recent modification of the method, this measurement is replaced by directly measuring cytotoxicity in the luciferase assay plates using the PRESTO BLUE reagent. This method had been validated on the draft OECD performance standards with equal results, but it has the advantage of increased statistical weight (triplicate analysis) and of measuring viability directly on the same cells as measuring luciferase

GLP compliance:

no

Remarks:

Conducted within the "spirit" of GLP (See Principles of method if other than guideline)

Type of study:

other: KeratinosensTM assay

Specific details on test material used for the study:

Trivial name : KEPHALIS
Chemical name : Cyclohexanone, 4-(1-ethoxyethenyl)-3,3,5,5-tetramethyl-
Molecular weight : 224.3
Molecular formula : C14H24O2
Purity : 80.9 % (sum of two main peaks)
Supplier : Givaudan Schweiz AG
Product code : 6378003
CAS number : 36306-87-3
EC number : 252-961-2
Expiration date : 19 Oct 2016
Batch number : PE00141817
Physical form : Liquid
Storage conditions : 4°C

Details on the study design:

Test System(s):
The KeratinoSens™ cell line is derived from the human keratinocyte culture HaCaT. It contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene AKR1C2 [2].
The KeratinoSens™ cell line was developed by the testing lab and stored on liquid nitrogen. It was grown in 10 cm petri dishes as described in the SOP to 80% confluency prior to testing for 3 – 4 days.
Cells were counted using a counting chamber and adjusted to the desired density. During seeding into 96-well plates, the cell suspension was gently stirred and cell sedimentation was avoided by repeatedly pipetting up and down to ensure homogeneous distribution of cells.

Basic Procedure:
Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a final level of 1% of the solvent DMSO containing the test substance. Each compound is tested at 12 concentrations in the range from 0.98 to 2000 μM. Each test plate contains six wells with the solvent control, 1 well with no cells for background value and 5 wells with a dose response of the positive control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same set-up.

Positive control
In each test Cinnamic aldehyde is included as positive control. It is tested in each test plate at five concentrations from 4 – 64 μM.

Endpoint & Endpoint Detection:
Two endpoints are measured: (i) Luciferase induction after a 48 h treatment with test substances and (ii) cytotoxicity as determined with the PRESTO BLUE assay [17].

Luminescence was read in a Promega Glomax Luminometer programmed to:
i. add 50 μl of the luciferase substrate to each well,
ii. to then wait for 1 second and
iii. then to integrate the luciferase activity for 2 seconds.

Endpoint Value:
For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5-, 2- and 3- fold induction (EC1.5, EC2 and EC3) are calculated. For cytotoxicity the IC50 value is extrapolated.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP. The test plates are read by a plate reader, and the generated raw data are directly pasted into this template, and all data processing is performed automatically by this Excel sheet.
For both the PRESTO BLUE and the luciferase data, first the background value recorded in an empty well without added cells is subtracted.
For the PRESTO BLUE data the % viability is then calculated for each well in the test plate in relation to average of the six solvent control wells.
For the luciferase data the average value of the six solvent control wells is set to 1, and for each well in the test plate the fold induction is calculated in relation to this value.

The following parameters are then calculated from these processed raw data:
• Imax Maximal fold-gene induction of the luciferase gene over the full dose-response
• EC 1.5 Concentration in μM for 1.5-fold gene induction
• EC 2 Concentration in μM for 2-fold gene induction
• EC 3 Concentration in μM for 3-fold gene induction
• Pos / Neg Rating of substance according to prediction model
• reps. Positive number of independent repetitions positive / number of repetitions done
• IC50 Concentration in μM for 50% reduction of cell viability

Prediction Model
Substances are rated positive if the following conditions are met::
• The Imax indicates > 1.5-fold gene induction, and this induction is statistically significant above the solvent control in a particular repetition as determined by students T-test. The EC1.5 value is below 1000 μM in all three repetitions or in at least 2 repetitions. (If the Imax is exactly equal to 1.5, the substance is still rated negative and no EC1.5 value is calculated by the evaluation sheet.)
• At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC 1.5 determining value), the cellular viability is above 70%.
• There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

Testing of Proficiency chemicals and historical positive control data in the test facility
The KeratinoSens assay was originally developed at the testing facility. Data for the Proficiency chemicals as defined by OECD TG 442d generated in the laboratory are summarized in the Givaudan report GCR 153’464 ’KeratinoSens assay: Proficiency testing at the testing facility’ [18].
The validation of the luciferase readings according to Annex 3 of the OECD guideline is also given in that report.
Results for repeated testing of the positive control are shown in Figure 6.
These validations show a very good reproducibility of the assay over time.

Run / experiment:

other: Rep 1

Parameter:

other: IC50 (μM)

Value:

186.79

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: Rep 2

Parameter:

other: IC50 (μM)

Value:

176.92

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: Rep 3

Parameter:

other: IC50 (μM)

Value:

168.88

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Geometric Mean IC 50 (μM)

Value:

177.38

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Standard deviation IC 50 (μM)

Value:

8.97

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 4. Cytotoxicity determinations. Given is the IC50 value as the concentration in μM reducing the viability by 50%.

Test Substance	Rep 1 IC50 (μM)	Rep 2 IC50 (μM)	Rep 3 IC50 (μM)	Geometric MeanIC50 (μM)	Standard deviationIC50 (μM)
Kephalis	186.79	176.92	168.88	177.38	8.97

Interpretation of results:

GHS criteria not met

Conclusions:

The result of the KeratinoSens™ assay should be used as part of an integrated approach for testing and assessment (IATA)[9]. A parallel test in the DPRA may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of substances, two congruent results in these two tests give a good prediction of the sensitizer hazard [5, 7, 19], in particular when comparing against human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.

In all three repetitions, no induction of the luciferase above the threshold of 1.5 was noted. Kephalis is thus rated negative in the KeratinoSens™ assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available