Reaction mass of 4-methyl-2-phenyl-3,6-dihydro-2H-pyran and 4-methyl-6-phenyl-3,6-dihydro-2H-pyran and 4-methylene-2-phenyltetrahydro-2H-pyran

Endpoint summary

Description of key information

Weight of Evidence: non-skin sensitising, 2019

1. Molecular initiating Key Event 1: negative (no to minimal peptide reactivity class), DPRA, OECD TG 442C, 2019

2. Molecular initiating Key Event 2: negative (no Luciferase activity induction > 1.5 in 2 of 2 experiments (n=3 replicates), KeratinoSens, OECD TG 442D, 2019

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:

28-11-2018 to 04-12-2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: January 2018 ; signature: June 2018

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (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 in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection and/or analysis. The test item achieved solubility in acetonitrile at a nominal concentration of 100 mM. Solutions of the test item in acetonitrile were successfully analysed by the validated DPRA analytical method (cited in the full study report) in both the Cysteine and Lysine containing synthetic peptides.
- HPLC-PDA (UV) methodology are reported in the full study report.
- Preparation of synthetic peptide solutions [Synthetic Peptide Containing Cysteine (SPCC) and Synthetic Peptide Containing Lysine (SPCL)]
1. Cysteine and Lysine: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca. 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).
2. Calibration: standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.
3. Stability control, Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report).
- Sample incubations: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were incubated, as applicable. The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.
- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = > 0.999 and SPLC r2 = > 0.999)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 72.6% SD: 0.15% and SPCL 59.2% SD: 0.89%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.15% and SPCL PC : SD = 0.89%)
(iv) mean peptide concentration of Reference Controls is to be 0.50 ± 0.05 mM (Actual: Cysteine: reference controls: 0.506 mM CV 0.34% ; Lysine: reference controls: 0.505 mM CV 0.33%, respectively).
(v) Coefficient of Variation (CV) of peptide areas for: Reference Controls in ACN are to be <15.0%. (Actual: Reference Controls: Cysteine: CV = 0.34% ; Lysine: CV = 0.33%)
- For each peptide analysis all acceptance criteria were met: linearity of standard response, reference and stability control concentrations, positive control depletion and test item reproducibility
- There was no co-elution of test item with either peptide.
- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=751) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=776) – full details on source provided in full study report.
- Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; 99.1%) – full details on source provided in full study report.
Negative control (NC): Vehicle = Acetonitrile

Evaluation of results: In accordance with OECD TG 442C – Table 1.
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity (Positive)
> 22.62 < 42.47 moderate reactivity (Positive)
> 6.38 < 22.62 low reactivity (Positive)
< 6.38 minimal reactivity (Negative)

Positive control results:

- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 72.6% SD 0.15% (high reactivity)
- PC LYS-peptide depletion (mean): 59.2% SD 0.89% (high reactivity)

Run / experiment:

other: Mean (%)

Parameter:

other: Cys-peptide depletion

Value:

0.605

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Test item appears to have no or minimal reactivity to CYS peptide

Remarks:

n = 3 ; See 'any other information on results incl. tables' for further information

Run / experiment:

other: Mean (%)

Parameter:

other: Lys-peptide depletion

Value:

-0.387

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Test item appears to have no or minimal reactivity to LYS peptide

Remarks:

n = 3 ; See 'any other information on results incl. tables' for further information

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: 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 in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = > 0.999 and SPLC r2 = > 0.999)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 72.6% SD: 0.15% and SPCL 59.2% SD: 0.89%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.15% and SPCL PC : SD = 0.89%)
(iv) mean peptide concentration of Reference Controls is to be 0.50 ± 0.05 mM (Actual: Cysteine: reference controls: 0.506 mM CV 0.34% ; Lysine: reference controls: 0.505 mM CV 0.33%, respectively).
(v) Coefficient of Variation (CV) of peptide areas for: Reference Controls in ACN are to be <15.0%. (Actual: Reference Controls: Cysteine: CV = 0.34% ; Lysine: CV = 0.33%)
- For each peptide analysis all acceptance criteria were met: linearity of standard response, reference and stability control concentrations, positive control depletion and test item reproducibility
- There was no co-elution of test item with either peptide.
All relevant acceptability criteria were met.

Table 1.0 – Acceptability of the DPRA

	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r2>0.99	60.8-100 (SD <14.9%)	0.45-0.55 mM (CV <15%)	SD <14.9%
	Lysine	r2>0.99	40.2-69.0 (SD <11.6%)	0.45-0.55 mM (CV <15%)	SD <11.6%
Achieved results	Cysteine	r2>0.999	72.6 (SD, 0.15%, n=3)	B: 0.506 mM (CV 0.34%, n=6)	SD 0.23% (n=3)
	Lysine	r2>0.999	59.2 (SD, 0.89%, n=3)	B: 0.505 mM (CV 0.33%, n=6)	SD 0.52% (n=3)

CV: Coefficient of Variation

SD: Standard deviation

 

Table 2.0 – Results of the DPRA with the test item

	Mean peak area of peptide	Mean peak area of peptide with test item (µV.sec)	Mean peptide depletion of test samples (%)
Cysteine	Control B: 920490 (n=6)	914920 (n=3)	0.605
Lysine	Control B: 797450 (n=6)	800540 (n=3)	-0.387

Interpretation of results:

other: The test item gave a negative and was classified in the “no to minimal reactivity class” using the Cysteine 1:10 / Lysine 1:50 prediction model. The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Conclusions:

Under the condition of this study, the test item is not considered to be sensitising to the skin. The test item indicated a negative in the DPRA and was classified in the “no to minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The study was performed to the OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either synthetic peptide cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and UV detection at 220 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Acetonitrile was found to be an appropriate solvent to dissolve the test item at up to nominal 100 mM concentration. In the cysteine reactivity assay the test item showed 0.605% SPCC depletion while in the lysine reactivity assay the test item showed -0.387% SPCL depletion. All relevant test acceptability criteria were met. Under the condition of this study, the test item is not considered to be sensitising to the skin. The test item indicated a negative in the DPRA and was classified in the “no to minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10-12-2018 to 10-01-2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-2 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-2 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: January 2018 ; signature: June 2018

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422D – In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method and EURL ECVAM DB-ALM Protocol no. 155: KeratinoSens™, (Adopted March, 2018).
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- A solubility test was performed. The test item was suspended or dissolved in DMSO to a final concentration of 200 mM. The 2000 µM (200 mM stock) concentration in DMSO was selected as highest concentration for the main assay.
- Test Item preparation and concentrations: In the main experiments the test item was suspended in dimethyl sulfoxide (DMSO) at 200 mM. From this stock, 12 concentrations were tested: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM, respectively. Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.
- Positive Control preparation and concentrations: The positive control was cinnamic aldehyde, for which a dilution series of: 4, 8, 16, 32 and 64 µM were prepared in DMSO. All concentrations of the positive control were tested in triplicate. Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.

- Acceptability criteria:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant and above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM). Actual PC: Experiment 1: 9.1 µM; Experiment 2: 6.96 µM.
(ii) The EC1.5 should be within the historical mean. Moreover, the induction for cinnamic aldehyde at 64 μM should be between 2 and 8. Actual PC average induction: Experiment 1: 4.76; Experiment 2: 6.60.
(iii) average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition (n=6, tested in triplicate): the % variability in solvent controls: Actual: Experiment 1: 6.3% ; Experiment 2: 7.8%.

Cell line used:
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. The cell line was developed by supplier (full details in the full study report). Cell cultures are maintained from frozen stocks. Cells can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for testing using the appropriate maintenance medium.

Cell culture and exposure:
Cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM), supplemented with 50 mL foetal bovine serum (FBS) and 5.5 mL Geneticin. Frozen culture was thawed in a water bath and resuspended in maintainece medium and further processed. The cells were sub-cultured up to 25-passages from frozen stock. Following further processing, the cell suspension was diluted with maintenance medium without geneticin to give 1 x 10^5 viable cells/mL and 100 μL volumes pipetted into all wells except one well of sterile 96-well flat-bottomed microtitre plates which received 100 μL maintenance medium without geneticin with no cells. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. The plates were incubated for 24 ± 2 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach. Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 μL of assay medium was added to every well of the 96 well plates. 50 μL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.

The following parameters are calculated in the KeratinoSens test method:
(i) The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
(ii) The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) is obtained
(iii) The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

- Cell viability assay MTT:
Test item IC50: IC50 value as the concentration in μM reducing the viability by 50%
Experiment 1: mean (n=3) 521.99 μM ; and Experiment 2: mean (n=3) 737.02 μM
Test item IC30: IC30 value as the concentration in μM reducing the viability by 30%
Experiment 1: mean (n=3) 404.73 μM ; and Experiment 2: mean (n=3) 625.89 μM

- Luciferase assay
Imax indicating maximum fold-induction up to concentration 2000 μM
Experiment 1: mean (n=3) 1.12 and Experiment 2: mean (n=3) 1.09

- Determinations:
EC1.5
Experiment 1: mean (n=3) could not be determined and Experiment 2: mean (n=3) could not be determined.

Evaluation criteria
Test item considered ‘negative’ where
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction (biphasic response)
Negative results obtained with concentrations <1000 µM or 200 µg/mL and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered as inconclusive.

Results:
2 out of 2 negative experiments (each in triplicate). The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 µM (dilution) for 48 hours ± 2 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The test item is classified as negative in the KeratinoSens assay since no positive results (>1.5-fold induction) were observed at test concentrations < 2000 µM with a cell viability of >70% compared to the vehicle control. The Imax for both tests was <1.5 fold compared to the DMSO control and therefore the EC1.5 could not be calculated. The cellular viability fell below 70% in both tests and no overall dose-response for induction could be established.

Positive control results:

All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant and above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM). Actual PC: Experiment 1: 9.1 µM; Experiment 2: 6.96 µM.
(ii) The EC1.5 should be within the historical mean. Moreover, the induction for cinnamic aldehyde at 64 μM should be between 2 and 8. Actual PC average induction: Experiment 1: 4.76; Experiment 2: 6.60.
(iii) average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition (n=6, tested in triplicate): the % variability in solvent controls: Actual: Experiment 1: 6.3% ; Experiment 2: 7.8%.

Historical results for luciferase induction by the positive control in the test laboratory: average and standard deviations from 30 valid runs are presented in the full study report.

Run / experiment:

other: mean Experiment 1 (n = 3)

Parameter:

other: EC1.5

Remarks:

/ µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: mean Experiment 2 (n=3)

Parameter:

other: EC1.5

Remarks:

/ µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: - The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant and above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM). Actual PC: Experiment 1: 9.1 µM; Experiment 2: 6.96 µM.
(ii) The EC1.5 should be within the historical mean. Moreover, the induction for cinnamic aldehyde at 64 μM should be between 2 and 8. Actual PC average induction: Experiment 1: 4.76; Experiment 2: 6.60.
(iii) average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition (n=6, tested in triplicate): the % variability in solvent controls: Actual: Experiment 1: 6.3% ; Experiment 2: 7.8%.

Interpretation of results:

other: The test item gave 2 out of 2 negative experiments (each in triplicate). The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Conclusions:

Under the condition of this study, the test item is not considered to be sensitising to the skin. The test item gave 2 out of 2 negative experiments (each in triplicate) where >1.5-fold induction was not observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

Executive summary:

The study was performed to the OECD TG 442D in vitro Skin Sensitisation guideline: ARE-Nrf2 Luciferase Test Method under GLP. The objective of this study was to evaluate the ability of the test item, to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in two independent experiments. The test item was suspended in dimethyl sulfoxide at 200 mM. From the stock solution, 100-fold solutions in DMSO were prepared for the assay resulting in test concentrations of 0.98 – 2000 µM. The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was within the historical mean (9.12 µM and 6.96 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 64 µM was higher than 2-fold in experiment 2 (4.76-fold and 6.60-fold in experiment 1 and 2, respectively). The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.3% and 7.8% in experiment 1 and 2, respectively). The test item showed toxicity (IC30 values of 404.73 µM and 625.89 µM and IC50 values of 521.99 µM and 737.02 µM in experiment 1 and 2, respectively). No dose-related induction of the luciferase activity could be measured in both experiments (EC1.5 values could not be determined). The maximum luciferase activity induction (Imax) was 1.12-fold and 1.09-fold in experiment 1 and 2. All relevant test acceptability criteria were met. The test item is classified as negative in the KeratinoSens assay since no positive results (> 1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of > 70% compared to the vehicle control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin Sensitisation:

1. Key Study – Molecular initiating Key Event 1: DPRA, OECD TG 442C, 2019 : The study was performed to the OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either synthetic peptide cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and UV detection at 220 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Acetonitrile was found to be an appropriate solvent to dissolve the test item at up to nominal 100 mM concentration. In the cysteine reactivity assay the test item showed 0.605% SPCC depletion while in the lysine reactivity assay the test item showed -0.387% SPCL depletion. All relevant test acceptability criteria were met. Under the condition of this study, the test item is not considered to be sensitising to the skin. The test item indicated a negative in the DPRA and was classified in the “no to minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

 

2. Key Study – Molecular initiating Key Event 2: KeratinoSens, OECD TG 442D, 2019 : The study was performed to the OECD TG 442D in vitro Skin Sensitisation guideline: ARE-Nrf2 Luciferase Test Method under GLP. The objective of this study was to evaluate the ability of the test item, to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in two independent experiments. The test item was suspended in dimethyl sulfoxide at 200 mM. From the stock solution, 100-fold solutions in DMSO were prepared for the assay resulting in test concentrations of 0.98 – 2000 µM. The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was within the historical mean (9.12 µM and 6.96 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 64 µM was higher than 2-fold in experiment 2 (4.76-fold and 6.60-fold in experiment 1 and 2, respectively). The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.3% and 7.8% in experiment 1 and 2, respectively). The test item showed toxicity (IC30 values of 404.73 µM and 625.89 µM and IC50 values of 521.99 µM and 737.02 µM in experiment 1 and 2, respectively). No dose-related induction of the luciferase activity could be measured in both experiments (EC1.5 values could not be determined). The maximum luciferase activity induction (Imax) was 1.12-fold and 1.09-fold in experiment 1 and 2. All relevant test acceptability criteria were met. The test item is classified as negative in the KeratinoSens assay since no positive results (> 1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of > 70% compared to the vehicle control.

 

Weight of Evidence Conclusion:

The applicant assesses the relevant studies by expert judgement and indicates that the weight of evidence indicates that there is no evidence of skin sensitisation in two validated in chemico (DPRA) and in vitro (KeratinoSens) skin sensitization assays covering Key Event 1 and Key Event 2 in the OECD IATA AOP for skin sensitization. The weight of evidence is indicative of an absence of significant skin sensitisation potential of the substance in accordance with the Regulation (EC) 1272/2008 criteria.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation.

The applicant assesses the relevant studies by expert judgement and indicates that the weight of evidence indicates that there is no evidence of skin sensitisation in two validated in chemico (DPRA) and in vitro (KeratinoSens) skin sensitization assays covering Key Event 1 and Key Event 2 in the OECD IATA AOP for skin sensitization. The weight of evidence is indicative of an absence of significant skin sensitisation potential of the substance in accordance with the Regulation (EC) 1272/2008 criteria.