α,2,2,6-tetramethylcyclohexene-1-butyraldehyde

Endpoint summary

Description of key information

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

In parallel, CETONAL was toxic to the KeratinoSens™ cells at 24.5 μM. It did induce the luciferase gene above a threshold of 1.5 only at a partly cytotoxic concentration of 17.6 μM in two of three repetitions. Since luciferase induction at cytotoxic concentrations only is rated negative according to the prediction model, CETONAL is considered a non-sensitizer according to the prediction model of the KeratinoSens™ assay.

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:

key study

Study period:

2015

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:

Test material name (as stated in the report): Cetonal
Batch No.: SC00008079
Expiry date: 22 June, 2015

Details on the study design:

BASIS OF THE METHOD
Skin sensitizing chemicals have the ability to covalently modify skin proteins or to be biotically or abiotically activated to become protein-reactive. Chemical-modified proteins are recognized by the immune system as foreign and trigger a specific T-cell mediated immune response. A key step in the skin sensitization process is therefore the formation of a covalent adduct between the skin sensitizer and endogenous proteins and/or peptides in the skin. Based on this wellestablished toxicity mechanism, the most straightforward approach to predict skin sensitization involves the measurement of the reactivity of a test compound towards peptides and proteins (reviewed in [7]). Gerberick et al. [2, 4] therefore developed a peptide depletion assay using different heptapeptides (later coined the DPRA or ‘direct peptide reactivity assay’) to assess a chemicals ability to react with and deplete a test peptide. Depletion is measured as the loss of the peptide signal as determined by HPLC-UV.

EXPERIMENTAL DESCRIPTION
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.

HPLC-Conditions:
▪ LC-System: Agilent 1100 Series (quat. low pressure gradient pump)
▪ Column: Zorbax SB-C18, 3μm, 2.1mm x 100mm
▪ DAD-Detector: 220nm
▪ Inj.Vol.: 7μl, Temp.: 30°C, Flow: 0.35ml/min
▪ Mobile Phase: ACN + 0.085% TFA / H2O + 1% TFA
▪ Gradient: 0min: 10% ACN / 90% H2O
10min: 25% ACN / 75% H2O
11min: 90% ACN / 10% H2O
13min: 90% ACN / 10% H2O
13.5min - 20min: 10% ACN / 90% H2O (conditioning)

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 is described in Test report RCR 153’453, ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the Givaudan testing facility’ [8].

Key result

Parameter:

other: Cys-peptide depletion

Value:

10.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Parameter:

other: Lys-peptide depletion

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Parameter:

other: Average depletion Cys-and Lys-peptide

Value:

5.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (3.4% and 0.1% SD, respectively).
The coelution controls indicated no co-elution with an UV-absorbing component.

The test substance gave 10.3% depletion of the Cys-peptide and 0.3% depletion of the Lys-peptide.

The average peptide depletion is 5.3 %. This is below the threshold of 6.38%, and the substance is thus attributed to the “minimal” reactivity class, rating it as a non-sensitizer according to the DPRA prediction model.

Interpretation of results:

GHS criteria not met

Conclusions:

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 substances, two congruent results in these two tests give a good prediction of the sensitizer hazard [3, 5, 9], while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.
CETONAL 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 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).

The test substance CETONAL 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 CETONAL was determined by HPLC-UV.

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

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

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:

The protocol was published as DB-ALM protocol 155 [1]. The assay was proposed by ECVAM [9] to be to be used aspart of an integrated approach for testing and assessment (IATA).

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: KeratinosensTM assay

Specific details on test material used for the study:

Test material name (as stated in the report): Cetonal
Batch No.: SC00008079
Expiry date: 22 June, 2015

Details on the study design:

BASIS OF THE METHOD
The only feature all skin sensitizers have in common is their intrinsic electrophilicity or their potential to be metabolically transformed to electrophilic chemicals. The signaling pathway with the repressor protein Keap1(Kelch-like ECH-associated protein 1) and the transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2), which binds to the antioxidant / electrophile response element (ARE / EpRE), is known to respond to electrophilic chemicals and it was found to be a valuable cellular endpoint to detect skin sensitizers in vitro [10]. This result was confirmed by independent laboratories [11-16].

EXPERIMENTAL DESCRIPTION
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, and a forth parallel plate is prepared for cytotoxicity determination.

Parameter:

other: Cytotoxicity determinations

Remarks:

Rep 1 IC50 (μM)

Value:

26.56

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Cytotoxicity determinations

Remarks:

Rep 2 IC50 (μM)

Value:

27.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Cytotoxicity determinations.

Remarks:

Rep 3 IC50 (μM)

Value:

19.31

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Cytotoxicity determinations

Remarks:

Geometric Mean IC 50 (μM)

Value:

24.19

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: Cytotoxicity determinations.

Remarks:

Standard deviation IC 50 (μM)

Value:

4.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table (i). Luciferase determinations. Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 μM.

	Test substance	Rep 1 IMAX (fold induction)	Rep 2 IMAX (fold induction)	Rep 3 IMAX (fold induction)	Average IMAX (fold induction)	Standard deviation IMAX (fold induction)
1	Cetonal	4.88*	3.33*	1.06	3.09	1.92

* Viability at the inducing concentration was < 70%

Table (ii). Luciferase determinations. Given is the EC1.5, EC2 and EC3 value as the concentration in μM inducing the luciferase activity 1.5-fold, 2-fold and 3-fold up to a concentration of 1000 μM.

	Test substance	Extrapolated value	Rep 1  (μM)	Rep 2 (μM)	Rep 3 (μM)	Geometric Mean (μM)	Standard deviation (μM)
1	Cetonal	EC 1.5	17.51 *	17.79 *	n.i.	17.64	0.20
1	Cetonal	EC 2	19.54 *	21.46 *	n.i.	20.48	1.36
1	Cetonal	EC 3	23.61 *	28.81 *	n.i.	26.08	3.68

n.i. no induction above a given threshold

* Viability at the inducing concentration was < 70%,

Table (iii). Overall rating of the test substance according to the prediction model and the number of positive repetitions.

	Test susbtance	Reps pos.	Overall rating
1	Cetonal	0 of 3	Negative

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, 18], 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.
CETONAL was toxic to the KeratinoSens™ cells at 24.5 μM. It did induce the luciferase gene above a threshold of 1.5 only at a partly cytotoxic concentration of 17.6 μM in two of three repetitions, as can be seen from the individual dose-response curves. Since luciferase induction at cytotoxic concentrations only is rated negative according to the prediction model, CETONAL is considered a non-sensitizer according to the prediction model of the KeratinoSens™ assay.

Executive summary:

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response.

This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to 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).

The test substance CETONAL was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions, each time in three replicates. After 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

CETONAL was toxic to the KeratinoSens™ cells at 24.5 μM. It did induce the luciferase gene above a threshold of 1.5 only at a partly cytotoxic concentration of 17.6 μM in two of three repetitions. Since luciferase induction at cytotoxic concentrations only is rated negative according to the prediction model, CETONAL is considered a non-sensitizer according to the prediction model of the KeratinoSens™ assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

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

In parallel, CETONAL was toxic to the KeratinoSens™ cells at 24.5 μM. It did induce the luciferase gene above a threshold of 1.5 only at a partly cytotoxic concentration of 17.6 μM in two of three repetitions. Since luciferase induction at cytotoxic concentrations only is rated negative according to the prediction model, CETONAL is considered a non-sensitizer according to the prediction model of the KeratinoSens™ assay.

Therefore the CETONAL does not meet the criteria to be classified as Skin sensitizer accoring to the CLP Regulation (EC) No. 1272/2008.