1,3,3-trimethylnorbornan-2-one

Endpoint summary

Description of key information

Skin sensitisation (in chemico): Weight of evidence. Test method according to the OECD 442C Guideline with GLP. Under the experimental conditions the test substance may be classified as not skin sensitizer according to DPRA skin sensitization test.

Skin sensitisation (in vitro): Weight of evidence. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test substance may be classified as not skin sensitizer using the KeratinoSensTM test method.

Skin sensitisation: Weight of evidence. integrated testing strategy (ITS). Based on the“2 out of 3-sens ITS” approach as described in annex 1 of OECD guidance ENV/JM/MONO(2016)29, two negative results obtained in the tests addressing two different key events (KEs) means that the test substance does not need to be classified as skin sensitizer.

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:

05 February 2018- 02 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Details on study design:

ITEM SOLVENT
Initial election of test item solvent: solubility of the test item in an appropriate solvent was assessed before performing the assay. The chosen solvent was acetonitrile.

TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, Louisville KY, USA; Ref. Ac RFAACAA-COOH; batch no 170809, 171229)
Lysine peptide (supplier RS Synthesis, Louisville KY, USA; Ref. Ac-RFAAKAA-COOH; batch no 170809, 161121)

CONTROLS
Positive control: Cinnamic Aldehyde (CAS 104-55-2; batch no MKBX8146V, Sigma Aldrich, purity 98.9%)
Co-elution control: test item in phosphate buffer (100 mM; pH 7.5) for cysteine peptide and ammonium acetate buffer (100 mM; pH 10.2) for lysine peptide.
Reference control A, B and C were prepared by adding 250 µL of acetonitrile to 750 µL of 0.667 mM Lysine peptide prepared in 100 mM Ammonium acetate buffer or 0.667 mM cysteine peptide prepared in 100 mM phosphate buffer.
Reference control A was used to verify the accuracy of the calibration curve for peptide quantification. Reference control A was injected in triplicates.
Reference control B was injected in triplicates each both in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
Reference control C was included in every assay run together with the samples in triplicates. They were used to verify that the solvent does not have any impact on the percent peptide depletion. The Reference Controls C was used to calculate Percent Peptide Depletion by the test item.

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item and positive control were dispensed using micropipette into clean and dry glass vials. 100 mM stock solution of test item and positive control (Cinnamic aldehyde) was prepared fresh, immediately before use.
Test item solution: Test item stock solution of 100 mM was prepared by dispensing 35.3 µL of test item into a vial and dissolved by adding 1964.7 µL of acetonitrile and mixed well.
Positive control solution: Positive control of 100 mM stock solution was prepared by dispensing 26.5 µL into a vial followed by adding 1973.5 µL of acetonitrile.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: Cysteine peptide Ac-RFAACAA-COOH, 0.667mM, 0.501 mg/mL: Using an electronic balance, Cysteine peptide was weighed and dissolved in appropriate volume of phosphate buffer.
Lysine solution: Lysine peptide Ac-RFAAKAA-COOH, 0.667mM, 0.518 mg/mL: Using an electronic balance, Lysine peptide was weighed and dissolved in appropriate volume of ammonium acetate buffer.

TEST SOLUTIONS
For each run, test item stock and positive control solutions were prepared before the start of sample preparation for analysis.
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
Test samples were prepared in triplicates for both peptides.
Both cysteine and lysine peptides were prepared on the same day once and remaining experiments peptides were tested individually.
All the replicates analysed in the same run were prepared with the same peptide stock solutions.

1 ml of each solution was prepared according to the following quantities:
Cysteine test solution (0.5 mM Peptide, 5 mM test chemical): 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µl of test chemical solution or solvent for Reference controls + 200 µl of acetonitrile.
Lysine test solution (0.5 mM Peptide, 25 mM test chemical): 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of test chemical solution or solvent for Reference controls.

Vials were capped, vortexed to mix and placed in (dark) at 25ºC for 24 hours. HPLC analysis of the batch of samples was started after 24 hours the test item addition to the peptide solution.

HPLC ANALYSIS
Apparatus:
Column: Agilent Zorbax SB-C18 2.1 mm X 100 mm X 3.5 micron
Detector: Photodiode Array detector or Fixed Wavelength Absorbance detector with 220 nm signal for quantification.
Calibration curve:
Six peptides standards solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared in 20% acetonitrile in phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide. The dilution buffer was also included as blank in the standard calibration curve.
Equilibration of the column: not specified.
Volume injected: 10 µl of each sample
Flow rate: 0.35 mL/min
Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
Duration of re-equilibration to the initial conditions between injections: Not specified.
Analysis sequence:
Two separate analysis sequences were prepared as below:
Analysis sequence 1: Calibration standards (Standard curve), Reference Controls A and Co-elution Controls.
Analysis sequence 2: Reference Controls B and sets of replicates of Reference Controls C, Positive Control and test item.
The first analysis sequence was timed to complete prior to the end of the 24 hour incubation and the second analysis sequence was timed to assure that the first sample starts 24 ± 2 hours after the test item was mixed with the peptide solution.
The difference between the first replicate analysis and the last replicate analysis was not more than 30 hours.

DEVIATIONS FROM OECD GUIDELINE: No.




DEVIATIONS FROM OECD GUIDELINE: No.
The column used has 2.7 µm particle size when the column described in the OECD 442C has 3.5 µm particle size. According to the guideline, the set-up parameters were adjusted to guarantee an appropriate elution and integration of the cysteine and lysine peptides. Proficiency substances recommended in the OECD guideline were performed in these conditions.

Positive control results:

The depletion mean rate was 71.18% for cysteine peptide and 45.60% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.0% for the lysine.

Key result

Run / experiment:

other: mean of 3 repetitions

Parameter:

other: %depletion in lysine peptide

Value:

1.38

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Mean of 3 repetitions

Parameter:

other: %depletion in cysteine peptide

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Mean of lysine and cysteine peptides

Parameter:

other: mean %depletion in peptides

Value:

0.69

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: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean peptide concentration of reference controls A was 0.47 mM for both cysteine and lysine peptides which are equal to 0.50± 0.05 mM.
- Acceptance criteria met for reference controls B and C: yes, the mean peptide concentration of the three Reference Controls C was 0.46 mM and 0.52 mM for cysteine and lysine peptides respectively which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the nine controls B and C was 1.46% and 5.67% for cysteine and lysine peptide which is lower than 15%.
- Acceptance criteria met for positive control: Yes, the depletion mean rate was 71.18% for cysteine peptide and 45.60% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine. The SD of the 3 repetitions was 1.64% (limit < 14.9%) for the percent cysteine depletion and 19.34% (limit <11.6%) for the percent lysine depletion. However, this criterion is considered valid as the third replicate of percent lysine depletion is an outlier based on the results of Grubb’s test and the mean percent lysine peptide depletion of the positive control is 45.60% which is within the range as suggested by OECD 442C test guideline.
- Acceptance criteria met for variability between replicate measurements: Yes, SD of the 3 repetitions of the test item for each peptide was 4.78% for cysteine and 8.32% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively.

Table 3: Mean and SD of Percent Peptide depletion for Cysteine and Lysine

For Cysteine peptide
Sample ID	Peak area of cysteine peptide	% peptide depletion	Mean % Peptide Depletion	SD
Cinnamic aldehyde-replicate 1	972449.0	71.35	71.18	1.64
Cinnamic aldehyde-replicate 2	925851	72.72
Cinnamic aldehyde-replicate 3	1036665	69.46
D027-01 replicate 1	3439003.0	-1.31	-0.64 (Considered as 0)	4.78
D027-01 replicate 2	3243768.0	4.44
D027-01 replicate 3	3565975	-5.05

For Lysine peptide
Sample ID	Peak area of Lysine peptide	% peptide depletion	Mean % Peptide Depletion	SD
Cinnamic aldehyde-replicate 1	1731217	57.01	45.60	19.34
Cinnamic aldehyde-replicate 2	1750371	56.53
*Cinnamic aldehyde-replicate 3	3089854	23.26
D027-01 replicate 1	4357614	-8.22	1.38	8.32
D027-01 replicate 2	3790534	5.86
D027-01 replicate 3	3765346	6.49

D027-01: 3,3-Dimethyl-8,9-Dinorbornan-2-one

* replicate 3 is an outlier

Table 4: Reactivity Class Classification of Test Item and Positive Control as per Cysteine 1:10/Lysine 1:50 Prediction Model

Sample ID/ Code	Name of the chemical	Mean % of Cysteine Peptide Depletion	Mean % of Lysine Peptide Depletion	Mean % peptide depletion	Reactivity class	DPRA prediction
CA	Cinnamic aldehyde	71.18	45.60	58.39	High Reactivity	Positive
D027-01	3,3-Dimethyl-8,9-Dinorbornan-2-one	0	1.38	0.69	No or Minimal Reactivity	Negative

Interpretation of results:

other: DPRA test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.

Conclusions:

The test item shows mean depletion of cysteine and lysine peptides of 0.69%, reflecting no or minimal reactivity and thus a negative prediction of DPRA skin sensitization test.

Executive summary:

A DPRA skin sensitization test was performed for test item as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item and Cinnamaldehyde (positive control) were prepared at 100 mM in acetonitrile as solvent. Reference controls A, B and C were prepared with acetonitrile in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item, positive control and reference controls were incubated for 24 hours ± 2 hours at 25°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, and then analyzed with HPLC to measure the peptide depletion. Test samples were prepared in triplicates for both peptides. All validity criteria were fulfilled. The mean of cysteine and lysine peptide depletion by the positive control was 58.39% which shows that it is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of depletion by the test item was 0.69% which shows that the test item has no or minimal reactivity on the skin sensitisation potential. Under the testing conditions, the test item was concluded as negative or non-sensitiser with no or minimal reactivity in Direct Peptide Reactivity Assay (DPRA).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 March 2018 – 27 March 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Details on study design:

REAGENTS AND MEDIA
-Fetal Bovine Serum (FBS): Gibco (Lot # 42Q8252K)
-DMSO: Sigma (Lot # STBF3447V)
-D-MEM: Gibco (Lot # 1922818, 1930096)
-DPBS: Sigma (Lot # 0000291179)
-Geneticin (G418): Gibco(Lot #1828468)
-Passive Lysis Buffer, 5x: Promega (Lot # 0000195524)
-Luciferase Assay System: Promega (Lot # 0000237533)
-Triple Express Enzyme: Gibco (Lot # 1868581)

-Culture and maintenance medium: D-MEM containing Glutamax and supplemented with 9.1% fetal bovine serum and 500 μg/mL geneticin, Penicillin 100 IU/mL and Streptomycin 100 μg/mL.
-Treatment medium: D-MEM supplemented with foetal bovine serum to a final concentration of 1% in the medium.
-Staining solution: MTT (5 mg/mL in DPBS) and 200 μL of treatment medium.

TEST SYSTEM
-Cells: KeratinoSens™ (Givaudan)
-Culture: cells were cultured at 37ºC, 5% CO2 in culture medium.
-Passage number: Not specified.

CONTROLS
-Positive control: cinnamaldehyde (CAS 104-55-2; Sigma, batch no MKBV4784V; purity 98.9%)
-Negative (solvent) control: DMSO

CELLS SEEDING.
-Cells suspension: On the days of experiment, the media were replaced with fresh medium without geneticin in the morning. Cells harvested for experiment were 80 – 90 % confluent on the day of seeding and never grown to full confluency. The cells were washed twice with DPBS (5 mL / flask), then 2 ml TrypLE Express Enzyme solution was added and flask kept back in to the incubator. After cells have detached (approx. 15 mins), cells were re-suspended in maintenance medium without Geneticin and centrifuged at 1000 rpm for 5 mins. Then the medium was aspirated and pellet was resuspended in fresh medium and the cells were adjusted to a density of 80,000 cells / mL in the maintenance medium. A volume of 125 μl of the cell suspension (containing 10,000 cells) was added to the wells B1-B12 and H1-H11 of 96 well plates. Treatment medium not containing cells was added in H12 well of 96 well plate and kept as blank control.
-nº of culture plates: four; three 96-well white assay plates (for luciferase assay) and one 96-well flat bottom transparent plate (for cytotoxicity, MTT assay).
-Cell density: 10e4 cells per well.
-Incubation: the seeded plates were incubated at 37 ± 1ºC in the presence of 5% CO2 for 24 ± 1h.

PREPARATION OF THE TEST ITEM AND CONTROL SUBSTANCES.
Positive control: A volume of 5.3 µL of Cinnamaldehyde was mixed with 194.7 µL of DMSO to prepare a stock concentration of 200 mM. A final concentration of 6.4 mM was prepared by further diluting 32 μL of the 200 mM stock solution in 968 μl of DMSO.
Negative control: DMSO was used as negative (solvent) control.
Test Item Preparation: The final concentration of 200 mM was prepared by adding 35.3 µL of test item to 964.7 µL of DMSO.
Preparation of 100X Master Plates:
-For Test Item: The 100 × DMSO master plate was prepared from 200 mM stock solution of the test item. Test item at final concentrations of 100 mM, 50 mM, 25 mM, 12.5 mM, 6.25 mM, 3.125 mM, 1.56 mM, 0.78 mM, 0.39 mM, 0.195 mM and 0.098 mM was prepared in DMSO by Serial (2 fold) dilution of 200 mM Stock solution. The Test Compounds was prepared fresh every time.
-For Control: Cinnamic aldehyde at stock concentration of 6.4 mM was serially diluted in DMSO to prepare further concentrations of 3.2 mM, 1.6 mM, 0.8 mM, 0.4 mM.
Preparation of intermediate stock (25 fold dilution in Treatment Medium): All the master concentrations will be further diluted 25-fold in treatment medium to get the intermediate concentration containing 4% DMSO in final volume.
Preparation of Working stock: A 4-fold dilution of all the concentration in treatment medium was made to get the working concentrations containing 1% DMSO in final volume. The 96 well plate containing the cells were replaced with 150 µL of treatment medium and 50 µL of each concentration was added to the desired wells and mixed.


APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
After the incubation period the medium was removed by aspiration and replaced with 150 μL DMEM-medium containing 1% FBS without Geneticin (Treatment medium). A volume of 50 μL of each concentration from the intermediate stock was added to the respective prelabelled wells (B1-B12 test item concentration ranging from 0.095 μM to 2000 μM, H1-H6 DMSO control, H7-H11 positive concentration ranging from 4 μM to 64 μM, H12 only medium without cells) and mixed. All the plates were covered with a sealing tape to avoid evaporation of volatile compounds and to avoid cross-contamination between wells by volatile compounds. The plates were incubated at 37±1°C in presence of 5 % CO2 for 48 ± 2 h in the CO2 incubator.

CONCENTRATIONS TESTED
Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
Positive control: 5 concentrations according to a geometric progression of ratio 2 from 4 to 64 µM.

REPLICATES: The study was composed of 3 independent repetitions. For each repetition the test item and the controls were replicated on three independent plates for the measurement of induction and one plate for the measurement of cytotoxicity.

LUCIFERASE ACTIVITY.
-Apparatus: luminometer. Molecular Devices. Flex station 3.
-Procedure: After the treatment exposure time, the supernatant was aspirated from the white assay plates and discarded. The cells were washed by adding 100 μL of DPBS and aspirated. To each well, 20 μL of 1x passive lysis buffer was added (care was taken to avoid formation of foam) and the cells were incubated at Room temperature for 20 mins. After incubation, 50 μL of Luciferase substrate was added manually to the plates containing cell lysates and mixed. The plates with the cell lysate and the luciferase substrate were placed in the luminometer for reading, programmed to integrate the luciferase activity for 1500 ms (1.5 Seconds).

CITOTOXICITY ASSESSMENT.
-Apparatus: Absorbance reader. Molecular Devices. Flex station 3.
-Procedure: For the cell viability measurement, the medium was aspirated and discarded from the transparent assay plates. A volume of 200 μL of treatment medium was added to the wells. In addition, a volume of 27 μl of a MTT solution (5mg/mL in DPBS) was added to the respective well of the transparent 96-well plate. The plate was protected from light while adding MTT solution. The plates were incubated at 37 ± 1ºC in presence of 5% CO2 for 4 hours. After incubation, the MTT medium was removed and the cells were lysed by adding 200 μL of 10 % SDS to the respective wells and incubated overnight. After overnight incubation, the plates were shaken for 10 mins and absorbance was read at 600 nm using photometer.

DEVIATIONS FROM OECD GUIDELINE: No.

Positive control results:

Repetition 1: The maximal average induction of luciferase activity (Imax) was 4.33 at a concentration of 64 μM. The mean value EC1.5 was 15.41 μM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 3.88 at a concentration of 64 μM. The mean value EC1.5 was 10.78 μM.
Repetition 3: The maximal average induction of luciferase activity (Imax) was 2.86 at a concentration of 64 μM. The mean value EC1.5 was 16.07 μM.

Key result

Run / experiment:

other: Repetition 1

Parameter:

other: Imax

Value:

1.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Repetition 2

Parameter:

other: Imax

Value:

1.43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Repetition 3

Parameter:

other: Imax

Value:

1.33

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was 16.6, 13.0 and 15.0 in each repetition which are less than 20%.

- Acceptance criteria met for positive control: yes, the luciferase activity induction was statistically significant above the threshold of 1.5 in at least one dose tested for each repetition. The average induction in the three replicates at 64 μM was 4.33, 3.88 and 2.86 in each repetition (Range 2 and 8). EC 1.5 values were 15.41, 10.78 and 16.07 in each repetition, which are between 7 and 30 μM.

Interpretation of results:

other: KeratinoSensTM test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442D.

Conclusions:

According to the vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens assay), the test item was found to be a non-sensitizer.

Executive summary:

The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours ± 1 hour at 37°C, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 1 plate (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control 1% DMSO were placed in the seeded plates and incubated for 48 hours ± 2 hours at 37°C, 5% CO2. 3 repetitions were performed on a different day with fresh stock solution. All validity criteria were fulfilled. The test item did not show any statistical significant induction above the threshold of 1.5 at any of the concentration over the solvent control. Under the same circumstances the positive control showed statistically significant induction over the solvent control confirming the sensitivity of the assay. Based on these results it is concluded that the test item is a non-sensitizer in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens assay).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation (in chemico): Weight of evidence. The DPRA skin sensitization test was performed for test item as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item and Cinnamaldehyde (positive control) were prepared at 100 mM in acetonitrile as solvent. Reference controls A, B and C were prepared with acetonitrile in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item, positive control and reference controls were incubated for 24 hours ± 2 hours at 25°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, and then analyzed with HPLC to measure the peptide depletion. Test samples were prepared in triplicates for both peptides. All validity criteria were fulfilled. The mean of cysteine and lysine peptide depletion by the positive control was 58.39% which shows that it is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of depletion by the test item was 0.69% which shows that the test item has no or minimal reactivity on the skin sensitisation potential. Under the testing conditions, the test item was concluded as negative or non-sensitiser with no or minimal reactivity in Direct Peptide Reactivity Assay (DPRA).

Skin sensitisation (in vitro): Weight of evidence. The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours ± 1 hour at 37°C, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 1 plate (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control 1% DMSO were placed in the seeded plates and incubated for 48 hours ± 2 hours at 37°C, 5% CO2. 3 repetitions were performed on a different day with fresh stock solution. All validity criteria were fulfilled. The test item did not show any statistical significant induction above the threshold of 1.5 at any of the concentration over the solvent control. Under the same circumstances the positive control showed statistically significant induction over the solvent control confirming the sensitivity of the assay. Based on these results it is concluded that the test item is a non-sensitizer in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens assay).

Skin sensitisation: Weight of evidence. integrated testing strategy (ITS). Based on the OECD Guidance Document on the reporting of defined approaches to be used within integrated approaches to testing and assessment for skin sensitization ((ENV/JM/MONO(2016)29), the “2 out of 3-sens ITS” approach as described in its annex 1 has been selected to allow the classification of the substance.

The combination of test methods used in this strategy covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E or the modified Myeloid U937 Skin Sensitisation Test (mMUSST)].

As there is no differential weighting of the individual test methods used and no predefined sequential order of testing, the order and information source from which data is obtained is not defined. Due to the higher complexity and resources needed (e.g. flow cytometer needed) to conduct the tests used for KE 3, the DPRA (KE1) and Nrf2-ARE-based tests (KE2) are usually conducted first.

Based on the current knowledge, information obtained from peptide reactivity, whether obtained from in chemico or in silico methods, seems to show the highest predictive power and may provide more weight to the overall assessment of skin sensitisation (Natsch et al., 2013, Urbisch et al., 2015).

According to this approach two concordant results addressing two different key events (KEs) indicate the sensitising potential, i.e. two positive results indicate a sensitiser, two negative results indicate a non-sensitiser.

Thus, the test substance does not need to be classified as skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is not classified for skin sensitization according to CLP Regulation no. 1272/2008.