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Description of key information

Klimish 1 KeratinoSens™ assay: Test report on HEXENYL-3-CIS ISOBUTYRATE dated on 27.01.2016_ Result non-sensitizer

Klimish Direct peptide reactivity assay (DPRA): Test report on Hexenyl-3-cisisobutyrate dated on 16.6.2016 _ Result non-sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to
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 : Hexenyl-3-Cis Isobutyrate
Expiration date : 03.03.2016
Batch number : Ve00318809
Physical form : Liquid
Details on 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).

The test substance HEXENYL-3-CIS-ISOBUTYRATE 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 HEXENYL-3-CIS-ISOBUTYRATE 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.

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:
32.7
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Lyspeptide depletion
Value:
0.8
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Average peptide depletion
Value:
16.7
Vehicle controls validity:
valid
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 (1.7% and 0.5% SD, respectively).
The co-elution controls indicated no co-elution with an UV-absorbing component.

When using HPLC-UV analysis only, HEXENYL-3-CIS-ISOBUTYRATE led to 32.7% depletion of the Cys-peptide. It was therefore considered reactive and classified into the LOW reactivity class according to the classical DPRA prediction model.

When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test chemical. At the same time the test chemical triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation rather than adduct formation. Thus, based on this more sophisticated and detailed analysis the test chemical is not directly peptide reactive and the assay does not indicate sensitization potential for Hexenyl-3-cis-isobutyrate.

Interpretation of results:
GHS criteria not met
Conclusions:
When using HPLC-UV analysis only, HEXENYL-3-CIS-ISOBUTYRATE led to 32.7% depletion of the Cys-peptide. It was therefore considered reactive and classified into the LOW reactivity class according to the classical DPRA prediction model.
When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test chemical. At the same time the test chemical triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation rather than adduct formation. Thus, based on this more sophisticated and detailed analysis the test chemical is not directly peptide reactive and the assay does not indicate sensitization potential for Hexenyl-3-cis-isobutyrate.
Executive summary:

When using HPLC-UV analysis only, HEXENYL-3-CIS-ISOBUTYRATE led to 32.7% depletion of the Cys-peptide. It was therefore considered reactive and classified into the LOW reactivity class according to the classical DPRA prediction model.

When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test chemical. At the same time the test chemical triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation rather than adduct formation. Thus, based on this more sophisticated and detailed analysis the test chemical is not directly peptide reactive and the assay does not indicate sensitization potential for Hexenyl-3-cis-isobutyrate.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to
Guideline:
other: 2014. EURL ECVAM Recommendation on the Keratinosens Assay
Qualifier:
according to
Guideline:
other: DB-ALM protocol 155: KeratinosensTM protocol
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:
Trivial name : Hexenyl-3-Cis Isobutyrate
Expiration date : 03.03.2016
Batch number : Ve00318809
Physical form : Liquid
Details on 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, and a forth parallel plate is prepared for cytotoxicity determination.

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 MTT assay recorded in a parallel plate with the same cell batch and made up with the same dilutions of the test substances.
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.

Parameter:
other: IC50 (μM)
Run / experiment:
Rep 1
Value:
362.6
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Given is the IC50 value as the concentration in μM reducing the viability by 50%.
Parameter:
other: IC50 (μM)
Run / experiment:
Rep 2
Value:
383.27
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Given is the IC50 value as the concentration in μM reducing the viability by 50%
Remarks:
.
Parameter:
other: IC50 (μM)
Run / experiment:
Rep 3
Value:
381.54
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Given is the IC50 value as the concentration in μM reducing the viability by 50%
Key result
Parameter:
other: Geometric Mean IC 50 (μM)
Value:
375.68
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Given is the IC50 value as the concentration in μM reducing the viability by 50%
Parameter:
other: IMAX (fold induction)
Run / experiment:
Rep 1
Value:
1.08
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 μM.
Parameter:
other: IMAX (fold induction)
Run / experiment:
Rep 2
Value:
1.2
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 μM.
Parameter:
other: IMAX (fold induction)
Run / experiment:
Rep 3
Value:
1.18
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 μM.
Parameter:
other: IMAX (fold induction)
Run / experiment:
Average
Value:
1.16
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Given is the Imax values indicating maximal fold-induction up to a concentration of 1000 μM.
Other effects / acceptance of results:
Positive and negative control:
Cinnamic aldehyde was run in all three repetitions. Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in all three repetitions.
The induction at 64 μM and the EC 1.5 for cinnamic aldehyde were also calculated. The targets are: (i) Average induction in the three replicates for cinnamic aldehyde at 64 μM should be between 2 and 8, and (ii) the EC 1.5 value should be between 7 μM and 30 μM. At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed here both criteria were fulfilled in all three repetitions. Thus all three repetitions were valid for the positive control.

As second performance criterion, the variability of the solvent control must be below 20%.All three repetitions were valid for the solvent control.

Cytotoxic activity of the test substance:

HEXENYL-3-CIS ISOBUTYRATE was moderately cytotoxic in the tested concentration range with an IC50 of 375.7 microM.

Luciferase induction by the test substances

HEXENYL-3-CIS ISOBUTYRATE did not induce the luciferase gene above the threshold of 1.5 at any concentration and is thus rated negative in the KeratinoSens™ assay.

Table 6. 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)

 
 

HEXENYL-3-CIS

ISOBUTYRATE

 EC 1.5  n.i.  n.i.  n.i.  n.i.  
 

HEXENYL-3-CIS

ISOBUTYRATE

 EC 2  n.i.  n.i.  n.i.  n.i.  
 

HEXENYL-3-CIS

ISOBUTYRATE

 EC 3  n.i.  n.i.  n.i.  n.i.  

n.i. no induction above a given threshold

Table 7. Overall rating of the test substance according to the prediction model and the number of

positive repetitions.

   Reps pos. Overall rating 
 HEXENYL-3-CIS ISOBUTYRATE  0 of 3  NEGATIVE
     
     

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.

Interpretation of results:
GHS criteria not met
Conclusions:
In all three repetitions, no induction of the luciferase above the threshold of 1.5 was noted.
According to the prediction model of the KeratinoSens™ assay, the test substance is rated as a non-sensitizer. This is also clearly supported by the analysis of the dose-response curve with overall no induction of the luciferase reporter gene to be observed.
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 HEXENYL-3-CIS ISOBUTYRATE 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.

HEXENYL-3-CIS ISOBUTYRATE was moderately toxic to the KeratinoSens™ cells with an IC50 of 376 μM. In all three repetitions, it did not induce the luciferase gene above a threshold of 1.5. It is therefore 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

According to the 2 Kilimish studies menzioned above, the Hexenyl-3-cis-isobutyrate does not show any sensitization potential.

Hence the GHS criteria are not met for any sensitization classification.