Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 17 and 22 May, 2016.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 442C , but not GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

no

Remarks:

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.

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

EC Number:

252-335-9

EC Name:

Ethyl 2,6,6-trimethylcyclohexa-1,3-ene-1-carboxylate

Cas Number:

35044-59-8

Molecular formula:

C12H18O2

IUPAC Name:

ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Physical appearance: colourless to pale yellow liquid
- Stored in a dry, well ventilated and dark location at room
temperature (i.e. 10–30°C)

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm.
Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

Preparation of test item and controls:
- Vehicle: acetonitrile (supplier: Acros Organic/batch number: 325730025), purity: 99.99%
- Positive control: Name :Trans-Cinnamaldehyde (CAS No.: 104-55-2 / 14371-01-9, Batch No. : STBB9109V / 101121271, Supplier : Sigma-Aldrich), purity: > 99%
- Co-elution control samples: In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.
- Reference control samples: All these control samples were prepared in triplicate and at the nominal concentration of 0.500 +/- 0.05 mM in the solvent (peptide solution cysteine or lysine). These samples were used to:
. reference control A: check the accuracy of the calibration curve for peptide quantification,
. reference control B: check the stability of the peptide during analysis,
. reference control C: check that the solvent did not impact the percentage of peptide depletion.
- Test item formulation preparation: The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetonitrile (supplier: Acros Organic/batch number: 325730025)) at 100 mM. This formulation was colorless limpid solution and was used just after its preparation.

Cysteine peptide:
. Peptide sequence : AC-RFAACAA.
. Peptide sequence synonyms : CysReact
. Molecular weight : 750.1 g/mol
. Supplier : Genscript Inc.(Piscataway, NJ, USA)
. Batch No. : 110639001092910ZW
. Storage condition : stored at -20°C
. Description : White powder
. Purity : 96.0%
. Expiry date : no data
The cysteine peptide solution was prepared in an aqueous phosphate buffer (pH 7.5) solution.

Lysine peptide
. Peptide sequence : Ac-RFAAKAA
. Molecular weight : 775.4
. Supplier : Genscript Inc. (Piscataway, NJ, USA)
. Batch No. : P17111206
. Storage condition : At -20°C
. Description : White powder
. Purity : 95.6%
. Expiry date : no data
The lysine peptide solution was prepared in an aqueous ammonium acetate buffer (pH 10.5) solution.

Study design:
The test item was tested in one run per peptide. The run was processed as described below.

- Preparation of the samples:
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.
.

- Reference control A and B samples preparation
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.

- Reference control C sample preparation
Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.
For the reference control C prepared with cysteine peptide: 50 μL of vehicle (acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reference control C prepared with lysine peptide: In parallel, 250 μL of vehicle (acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.5).

- Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide: 5 mM with the Cysteine peptide was tested.
For the reactivity of cinnamaldehyde with lysine peptide: 25 mM with the Lysine peptide was tested.

- Test item samples preparation
For the reactivity of test item with cysteine peptide: 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 (5mM) dissolved in acetonitrile.
For the reactivity of test item with lysine peptide: 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.

- Incubation of the samples
All samples (co-elution control, reference controls, test item and positive control samples) were incubated during 24 hours before injection onto the HPLC/UV system. At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation.

- Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking both peptides (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.1 to 0.6 mM. A dilution buffer blank was also included in the standard calibration curve. The calibration curves were defined by the relationships between the peak area of the peptide signal versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

- HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis. For each peptide, the analytical sequence included at least:
. one blank sample (peptide dilution buffer),
. one calibration curve injected at the beginning of the analytical batch,
. three reference control A samples,
. one co-elution control sample,
. three reference control B samples,
. reference control C sample (replicate 1),
. positive control sample (replicate 1),
. test item study samples (replicate 1).
The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3:
. three reference control B samples.

The HPLC/UV method used for the samples analysis according to the recommendations of the OECD guideline No. 442C and is summarized in the table below:
. LC-System: Agilent 1100 series (quat. Low pressure gradient pump)
. Column: Zorbax SB-C18, 3 µm, 2.1 mm x 100 mm
. DAD-Detector: 220 nm
. Inj.Vol: 7 µL, Temp: 30°C, Flow: 0.35 ml/min
. Mobile Phase: CAN + 0.085% TFA/H2O +1% TFA.
. Gradient
0 min: 10% ACN / 90% H2O
10 min: 25% ACN / 75% H2O
11 min: 90% ACN / 10% H2O
13 min: 90% ACN / 10% H2O
13.5 min – 20 min: 10% ACN / 90% H2O (conditioning)

Results and discussion

Positive control results:

Cinnamic aldehyde was used as a positive control.

- Result of positive control samples in cysteine assay: Mean % depletion = 76.5% and SD = 0.4%
- Result of positive control samples in lysine assay: Mean % depletion = 57.5% and SD = 2.0%
Mean depletion rate of Cinnamaldehyde: 67%
The mean percent peptide depletion values for the positive control with its standard deviation value were within the acceptability criteria for the DPRA assay (cysteine and lysine reactivity assays).
All the acceptance criteria were fulfilled for the positive control cinnamic aldehyde, with the exception of the CV for the Lys peptide which is at 12.4%, and thus slightly above
the threshold of 11.6. However, since this is so close to the threshold and the three individual
depletion values (64.6, 50.9, 62.6) all nicely fall in the target range and the historical range, this was considered an acceptable deviation.

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: Mean % depletion in Cysteine assay

Value:

4.6 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Parameter:

other: Mean % depletion in Lysine assay

Value:

0.3 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Parameter:

other:

Remarks:

Mean depletion rate

Value:

2.5 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Minimal reactivity

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference controls: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Acceptance criteria met for the calibration curve: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

Any other information on results incl. tables

Solubility results

The test item was found soluble at 100 mM in acetonitrile without sonication step. Therefore, this vehicle was retained.

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item 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 skin sensitisation potential of the test item was evaluated using an in chemico direct peptide binding assay (DPRA) in accordance to the OECD Guideline No. 442C and not in compliance with GLP. The reactivity of the test item was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item at the ratios 1:10 cysteine:test item and 1:50 lysine:test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with ultra-violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

The test item was dissolved at 100 mM in acetonitrile without sonication.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

. for the cysteine peptide, the mean depletion value was 4.6%,

. for the lysine peptide, the mean depletion value was 0.3%.

The mean of the percent cysteine and percent lysine depletions was equal to 2.5%. This is below the threshold of 6.38%, and the test item was considered have minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test item may have no potential to cause skin sensitization.

Under the experimental conditions of this study, the test item 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.