3,7-dimethyloct-1-en-3-yl acetate

Endpoint summary

Description of key information

The test item DMOE Acetate was negative in the DPRA (OECD 442C) and in the ARE-Nrf2 Luciferase Test (KeratinoSens, OECD 442D). Thus, the test item was judged to be not skin sensitising.

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

Study period:

15 June 2015 to 29 June 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 Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
Co-elution control samples preparation
For the co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

Reference control samples preparation
Reference control A and B samples: 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 samples
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 10.2).

Cinnamaldehyde (positive control) depletion control samples preparation
For the reactivity of cinnamaldehyde with cysteine peptide:
50 μL of cinnamaldehyde at 100 mM in 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 reactivity of cinnamaldehyde with lysine peptide:
In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Test item samples preparation
For the reactivity of test item with cysteine peptide:
50 μL of test item formulation 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 reactivity of test item with lysine peptide:
In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).


INCUBATION
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours with cysteine peptide or during approximately 27 hours with lysine peptide at 25°C and protected from light 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.
As precipitates and/or micelles were observed in the co-elution and test item samples incubated with the cysteine and lysine peptides, these vials were centrifuged at 400 g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Only supernatants were then injected onto the HPLC/UV system.
Although precipitates were observed in the reference and positive control samples incubated with the cysteine and lysine peptides, these vials were not centrifuged at 400 g for a period of 5 minutes at room temperature since precipitates were already observed at the bottom of the vial. Only supernatants were injected onto the HPLC/UV system.


DATA EVALUATION
The study samples were assayed in batches using HPLC/UV analysis. The concentration of cysteine or lysine peptide was photometrically determined at 220 nm.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

The positive control depletion values for cysteine and lysineldepletion are within the ranges of the acceptance criteria.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes
- Acceptance criteria met for variability between replicate measurements: yes

 

Since precipitates and/or micelles were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity cannot be drawn with sufficient confidence. However, precipitates were observed in the positive control samples as well.

 

Table 1: Peptide Depletion

Sample	Cysteine peptide depletion (%)	Lysine peptide depletion (%)	Mean Depletion (%)
Positive control	72.24	54.92	63.20
	73.14	53.92
	71.20	53.78
DMOE-Ac	-2.60	-1.39	0
	-1.98	-0.90
	-3.47	-1.56

 

Interpretation of results:

other: This study alone is not sufficient to decide on the classification of a substance as skin sensitiser.

Conclusions:

The negative Direct Peptide Reactivity Assay (DPRA)-result can be used as part of a testing battery (including e.g. h-CLAT (human cell line activation test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.

Executive summary:

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 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. 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. For both peptides, the mean depletion value was set to 0 due to negative percentage depletion value. The mean of the percent cysteine and percent lysine depletions was therefore equal to 0%. Accordingly, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction would be considered as negative. Since precipitates and/or micelles were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity cannot be drawn with sufficient confidence. However, precipitates were observed in the positive control samples as well.

As a conclusion, under the experimental conditions of this study, the test item Dimethyl Octenyl Acetate (DMOE-Ac) was considered to have no/minimal peptide reactivity, though with limitations due to presence of precipitates and/or micelles at the end of the incubation with peptides in test item samples.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 May 2015 to 05 June 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:

February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
On the basis of solubility results, the test item was dissolved in DMSO at 200 mM.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2, to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called Master plate 4x taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.
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 in the Master plate 100x, using a dilution factor of two, to obtain a total of five concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called Master plate 4x. The final tested concentrations ranged from 4 to 64 μM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.
Vehicle and negative control:
Based on solubility results, the vehicle was DMSO.
This vehicle was used as the negative control, and was applied to cells at a concentration of 1% in culture medium.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: controls: 3
- Number of repetitions: 2
- Test chemical concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM
- Application procedure
Treatment
- After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium,
- from the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation,
- all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
- the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

Study evaluation and decision criteria used
Acceptance criteria
Each run was considered valid if the following criteria were met:
- the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within 7 and 30 μM. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between two and eight. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control,
- the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%. For the first run, an outlier luminescence value (first replicate of the second plate) was removed from the data analysis, therefore this acceptance criteria was based on 17 values instead of 18.

Evaluation criteria
The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- the EC1.5 value is < 1000 μM (or < 200 μg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).

Luminescence flash signal to evaluate induction signal - white plates
- After incubation, the supernatants from the white assay plates were discarded,
- the cells were washed once with D-PBS,
- a volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking,
- the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
- 50 μL of the luciferase substrate was added to each well,
- 1 second after this addition, the luciferase signal was integrated for 2 seconds-

Absorbance signal to evaluate the cytotoxicity - transparent plate
- For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium,
- a volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
- the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes),
- at the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well,
- the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
- after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

Vehicle / solvent control:

DMSO

Negative control:

other: The vehicle (DMSO) was used as negative control.

Positive control:

cinnamic aldehyde [442D]

Positive control results:

All acceptance criteria were met for the positive and negative controls in both runs, they were therefore considered as valid.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

50.9 µM

Cell viability:

A decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 62.5 μM in both runs.

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

same as vehicle control

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

43.8 µM

Cell viability:

a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 62.5 μM in both runs.

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

same as vehicle control

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

No statistically significant gene-fold induction was noted in comparison to the negative control at any tested concentrations in either run. Moreover, the Imax values were ≤ 1.5 (mean Imax value for both runs: 1.36).

The evaluation criteria for a negative response are met in both 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 skin sensitisation potential.

First run

With one exception which was considered not to have any impact on the validity of the results, all acceptance criteria were fulfilled and the run was therefore considered to be valid. In this run an outlier luminescence value was removed from the data analysis of the negative control wells (the first replicate of the second plate), therefore 17 instead of 18 values were taken into consideration for the analysis.

This run was performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO.

At these tested concentrations:

- a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 62.5 μM,

- the corresponding IC30 and IC50 were calculated to be 45.6 and 50.7 μM, respectively,

- no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was ≤ 1.5

 

Second run

With one exception which was considered not to have any impact on the validity of the results (see § Study plan adherence), all acceptance criteria were fulfilled and the run was therefore considered to be valid. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between two and eight" was not fulfilled (i.e. Imax of 9.5). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.

The same concentrations as those in the run 1 were used.

At these tested concentrations:

- a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 62.5 μM,

- the corresponding IC30 and IC50 were calculated to be 42.0 and 51.2 μM, respectively,

- no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was ≤ 1.5.

 

The geometric means IC30 and IC50 of the two runs were calculated to be 43.8 and 50.9 μM, respectively.

The evaluation criteria for a negative response are met in both 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 skin sensitisation potential.

 

Table 1: Imax, IC30, IC50 and EC1.5 values, mean and SD values obtained after treatment with the test item in each run

Test item	Imax	EC1.5 [µM]	IC50 [µM]	IC30 [µM]
Run 1	1.46	-	50.70	45.62
Run 2	1.26	-	51.15	41.96
Mean	1.36	n.r.	n.r.	n.r.
Geometric mean	n.r.	-	50.93	43.75
SD	0.14	-	0.32	2.59

 

Table 2: Imax, IC30, IC50 and EC1.5 values, mean and SD values obtained with the positive control for each run

Cinnamic aldehyde	Imax	EC1.5 [µM]	IC50 [µM]	IC30 [µM]
Run 1	5.38	3.94
Run 2	9.46	6.48
Mean	7.42	n.r.	n.r.	n.r.
Geometric mean	n.r.	5.05
SD	2.89	1.80

 

 

Interpretation of results:

other: The study results alone are not sufficient to decide on the classification for skin sensitisation.

Remarks:

The KeratinoSens test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay) and human cell line activation test method (h-CLAT)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Conclusions:

Under the experimental conditions of this study, the test item, Dimethyl Octenyl Acetate (DMOE-Ac), was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The objective of this study was to evaluate the potential of the test item, Dimethyl Octenyl Acetate (DMOE-Ac), to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential.

This in vitro test uses Human adherent HaCaT keratinocytes, an immortalized cell line. The KeratinoSens is a stably transfected cell line with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The KeratinoSens cells were plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control and to several concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed. For each run, the test item was solubilised in DMSO at 200 mM.

With one exception in each run which were considered not to have any impact on the validity of the results, all acceptance criteria were met for the positive and negative controls in both runs, they were therefore considered as validated.

Both runs were performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. At these tested concentrations:

- a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 62.5 μM in both runs,

- the geometric means IC30 and IC50 of the two runs were calculated to be 43.8 and 50.9 μM, respectively,

- no statistically significant gene-fold induction was noted in comparison to the negative control at any tested concentrations in either run. Moreover, the Imax values were ≤ 1.5.

The evaluation criteria for a negative response are met in both 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. Under the experimental conditions of this study, the test item, Dimethyl Octenyl Acetate (DMOE-Ac), 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:

The chemical and biological mechanisms associated with skin sensitisation are summarised in the form of an Adverse Outcome Pathway (AOP). This AOP includes four key events:

1) The molecular initiating event is the covalent binding of electrophilic substances to nucleophilic centres in skin proteins.

2) The inflammatory responses and gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways in the keratinocytes.

3) The activation of dendritic cells, typically assessed by expression of specific cell surface markers, chemokines and cytokines.

4) The T-cell proliferation.

Studies addressing one of the key events can be used as part of a testing battery based on the OECD adverse outcome pathway (AOP) for the assessment of the skin sensitisation potential of chemicals. For DMOE acetate, Key event 1 is addressed by the DPRA assay. The second key event is addressed by the KeratinoSens test. As the results of both tests were negative, no further testing is required.

 

DPRA Assay

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 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. 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. For both peptides, the mean depletion value was set to 0 due to negative percentage depletion value. The mean of the percent cysteine and percent lysine depletions was therefore equal to 0%. Accordingly, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction would be considered as negative. Since precipitates and/or micelles were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated. Therefore, the conclusion on the lack of reactivity cannot be drawn with sufficient confidence. However, precipitates were observed in the positive control samples as well.

As a conclusion, under the experimental conditions of this study, the test item Dimethyl Octenyl Acetate (DMOE-Ac) was considered to have no/minimal peptide reactivity, though with limitations due to presence of precipitates and/or micelles at the end of the incubation with peptides in test item samples.

 

KeratinoSens Assay

The objective of this study was to evaluate the potential of the test item, Dimethyl Octenyl Acetate (DMOE-Ac), to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential.

This in vitro test uses Human adherent HaCaT keratinocytes, an immortalized cell line. The KeratinoSens is a stably transfected cell line with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The KeratinoSens cells were plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control and to several concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed. For each run, the test item was solubilised in DMSO at 200 mM.

With one exception in each run which were considered not to have any impact on the validity of the results, all acceptance criteria were met for the positive and negative controls in both runs, they were therefore considered as validated. Both runs were performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. At these tested concentrations:

- a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 62.5 μM in both runs,

- the geometric means IC30 and IC50 of the two runs were calculated to be 43.8 and 50.9 μM, respectively,

- no statistically significant gene-fold induction was noted in comparison to the negative control at any tested concentrations in either run. Moreover, the Imax values were ≤ 1.5.

The evaluation criteria for a negative response are met in both 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. Under the experimental conditions of this study, the test item, Dimethyl Octenyl Acetate (DMOE-Ac), was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.

Conclusion

The study results are evaluated in a weight-of-evidence approach. As both results are negative, it is concluded that the test item DMOE-Acetate is not sensitising to skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on the results obtained, the test item was not classified for skin sensitisation according to Regulation (EC) No 1272/2008, as amended for seventeenth time in Regulation (EU) No 2021/849.