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

Endpoint summary

Description of key information

- In vitro study: covalent interaction with protein in skin (DPRA, OECD 442C, K, Rel.2): predicted to be negative in the DPRA assay.

- In vitro study: activation of keratinocytes (KeratinoSens, OECD 442D, K, Rel.2): predicted to be negative in the KeratinoSens assay.

- Repiratory sensitisation: no data available

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:

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.

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.

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)

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.

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

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

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

Solubility results

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

 

 

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 14 and 24 June, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 442D but Not GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

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 this study, the measurement is replaced by directly measuring cytotoxicity.

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:

activation of keratinocytes

Justification for non-LLNA method:

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

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Test item formulations/Treatment:
The test substance was freely soluble in DMSO at 200 mM, and this preferred solvent according the SOP could thus be used.
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.
Test System:
- KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test
- Supplier: this cell line was provided by Givaudan
- Batch: no data
- Storage condition: stored on liquid nitrogen.
- Mycoplasm: no data

Test reagents:
All test reagents were sourced as indicated in the standard operating procedure. Luciferin was sourced from Promega. The Luciferase substrate was prepared according to the following recipe: 20 mM Tricine; 2.67 mM MgSO4; 0.1 mM EDTA; 33.3 mM DTT; 270 µM Coenzyme A; 470 µM Luciferin; 530 µM ATP; pH 7.8
Cell seeding for testing:
- Cells were grown using general culture procedures up to 80% confluence, 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.
- 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.

Preparation of the positive control:
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions. It is tested in each test plate at five concentrations from 4 – 64 µM.
Endpoint measurements:
- Microscopic observation
After the 48 hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
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. Luminescence was read in a Promega Glomax Luminometer programmed to
i. add 50 µl of the luciferase substrate to each well,
ii. ii. to then wait for 1 second and
iii. iii. then to integrate the luciferase activity for 2 seconds.

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.
Cytotoxicity assessment using the PRESTO BLUE reagent:
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.
Data analysis:
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 up to 1000 µM.
- 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

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

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 (See Table 7.4.1/1). 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.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Remarks:

with EC1.5 calculated = 228.27 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Value: 1.57. Induction occurred at cytotoxic levels only, hence this repetition is rated negative.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Remarks:

no EC1.5 calculated

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Value: 0.89. No indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Remarks:

no EC1.5 calculated

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Value: 0.92. No indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Cell viability: the test substance was weakly toxic to the KeratinoSens cells in the tested concentration range (See table 7.4.1/2)
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 7.4.1/1: Numerical results for the positive control cinnamic aldehyde.

Quality control: Induction values Reference						Criteria
cinnamic aldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC 1.5	Ind. 64 µM	EC 1.5
Rep 1	1.15	1.29	1.75	2.25	3.37	11.62	True	True
Rep 2	1.36	1.57	1.62	2.13	4.02	21.37	True	True
Rep 3	1.13	1.42	1.63	2.53	3.42	11.16	True	True
aVERAGE	1.22	1.43	1.66	2.30	3.61	14.72

 

Table 7.4.1/2 . Cytotoxicity determinations. Given is the IC50 value as the concentration in µM reducing the viability by 50%

	Rep 1 IC50 (µM) (*)	Rep 2 IC50 (µM)	Rep 3 IC50 (µM)	Geometric Mean IC 50 (µM)	Standard deviation IC 50 (µM)
Test substance	316.56	313.48	309.28	313.10	3.653938

(*) The three values given come from three independent experiments

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The test item was evaluated for the ability to activate the Nrf2 transcription factor in a KeratinoSens(TM) test similar to the OECD Guideline No. 442D but not in compliance with GLP. In this test, KeratinoSens(TM) cells cultures were treated with 12 concentrations of test chemical (0.98, 1.95, 3.91, 7.81, 15.63, 62.5, 125, 250, 500, 1000 and 2000 µM) and control substances (positive control: Cinnamic Aldehyde in DMSO, vehicle control: 1% DMSO in exposure medium, blank) and were then incubated for about 48 hours at 37°C in presence of 5% CO2. Luciferase activity (Imax and EC1.5) was assessed using a luminometer. The toxicity of the test substance was evaluated with the PRESTO BLUE assay. A total of 3 independent experiments were performed.

 

All acceptance criteria were met for the positive and negative controls in all runs; they were therefore considered as validated.

 

The test substance was weakly toxic to the KeratinoSens cells in the tested concentration range.

The test substance did induce the luciferase gene above the threshold of 1.5 in one of three repetitions and at cytotoxic level only. The test substance is thus rated negative in the KeratinoSens™ assay. The Imax values were 1.57, 0.89 and 0.92 during the first, second and third runs, respectively. This conclusion is also clearly supported by the overall dose-response curve from the three repetitions.

 

The evaluation criteria for a negative response are met in the three runs, the final outcome is therefore negative. This negative result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a lack of skin sensitization potential.

 

Under the experimental conditions of this study, the test item was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Two in chemico/in vitro key studies were identified :

- In the in chemico assay (DPRA, Givaudan, 2016, rel.2) considered as the key study, the skin sensitisation potential of the test item was evaluated using a direct peptide binding assay (DPRA) similarly to the OECD Guideline No. 442C but 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.

 

- In the in vitro assay (KeratinoSens, Givaudan, 2016, Rel.2), considered as the key study, the test item was evaluated for the ability to activate the Nrf2 transcription factor in a KeratinoSens(TM) test similar to the OECD Guideline No. 442D but not in compliance with GLP. In this test, KeratinoSens(TM) cells cultures were treated with 12 concentrations of test chemical (0.98, 1.95, 3.91, 7.81, 15.63, 62.5, 125, 250, 500, 1000 and 2000 µM) and control substances (positive control: Cinnamic Aldehyde in DMSO, vehicle control: 1% DMSO in exposure medium, blank) and were then incubated for about 48 hours at 37°C in presence of 5% CO2. Luciferase activity (Imax and EC1.5) was assessed using a luminometer. The toxicity of the test substance was evaluated with the PRESTO BLUE assay. A total of 3 independent experiments were performed.

All acceptance criteria were met for the positive and negative controls in all runs; they were therefore considered as validated.

The test substance was weakly toxic to the KeratinoSens cells in the tested concentration range.

The test substance did induce the luciferase gene above the threshold of 1.5 in one of three repetitions and at cytotoxic level only. The test substance is thus rated negative in the KeratinoSens™ assay. The Imax values were 1.57, 0.89 and 0.92 during the first, second and third runs, respectively. This conclusion is also clearly supported by the overall dose-response curve from the three repetitions.

 

Based on the results of these in chemico/in vitro tests, the test item cannot be considered as a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available information, the substance should not be classified as skin sensitizer according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS.