2-Propenoic acid, 2-hydroxyethyl ester, reaction products with O,O-bis(dialkyalkyl) hydrogen phosphorodithioate and dimethyl phosphonate

Endpoint summary

Description of key information

In vitro

 

ARE-Nrf2 Luciferase Test (KeratinoSens)

The test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens), supporting the prediction that the test item is likely to be a skin sensitiser (OECD 442D).

 

Direct Peptide Reactivity Assay (DPRA)

The test item gave an overall mean peptide depletion rate of 1.60 % equivalent to 'no or minimal reactivity' and was considered unlikely to be a skin sensitiser (OECD 442C).

 

In vivo

 

Buehler method

The test item is not considered to be a contact sensitiser (OECD 406, EU Method B.6, OPPTS 870.2600 and relevant Japanese guidelines)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2019 to 26 September 2019

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:

June 2018

Deviations:

yes

Remarks:

deviations with respect to incubation time and cell confluency with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

PURPOSE OF STUDY
- The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens).
- The KeratinoSens in vitro skin sensitisation assay uses an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test chemicals.

CELL CULTURE
- The cells used in this assay were the transgenic cell line KeratinoSens with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland).
- The 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) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (10500) and 5.5 mL Geneticin (Gibco 10131).

CELL CULTURE FROM FROZEN STOCKS
- Vials of KeratinoSens cells, stored frozen in cryotubes at -196 °C under liquid nitrogen, in DMEM containing 10 % dimethyl sulphoxide and 20 % FBS, were thawed rapidly at 37 °C in a water-bath.
- The cells were then resuspended in 9 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes.
- The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks.
- The flasks were incubated until 80 to 90 % confluent cell monolayers had been obtained.
- Geneticin-containing medium was used in subsequent passages.
- Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of the study.

CELL PASSAGE
- Actively growing cell stocks were maintained and expanded by subculturing (passage).
- When the cells had reached 80 to 90 % confluence, the medium from each flask was removed, the cells washed twice with Dulbecco's phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution.
- Cultures were incubated at 37 ± 2 °C in a humidified atmosphere containing 5 % CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred.
- The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks were pooled at this point).
- The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test and were passaged at least twice before using the cells in a test. The passages of KeratinoSens cells were limited to 25 passages from frozen stock.

PREPARATION OF TEST CELL CULTURES
- The cells from flasks of actively growing cultures were detached and disaggregated as described above.
- The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer.
- The cell suspension was diluted with maintenance medium without geneticin to give 1 x 10E+05 viable cells/mL and 100 µL volumes pipetted into all wells except one well of sterile 96-well flat bottomed microtitre plates.
- 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. One well of each plate received 100 µL maintenance medium without geneticin with no cells.
- 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.

PREPARATION OF POSITIVE CONTROL
- Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 to 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using the formula V = 5 * [(p / 100) * w) / MW] - (w / 1000) where V = volume of DMSO in mL to be added; p = purity of chemical in %; MW = molecular weight of the chemical in g/moL; w = exact weight of the chemical added to the vial in mg.
- The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.
- Preparation of the positive control was shared with other studies performed in the same assay.

TEST ITEM SOLUBILITY
- Prior to commencing testing, the solubility of the test item in assay medium containing 1 % DMSO at 200 mM was assessed.
- The test item was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.

PREPARATION OF THE TEST ITEM
- A stock solution of the test item was prepared by weighing the test item into a tared glass container and diluting to 200 mM in DMSO.

PREPARATION OF THE 100x SOLVENT PLATE
- A 100x solvent plate was set up by adding 200 µL of the stock solution of the test item to one well in column 12.
- A 200 µL volume of the 6.4 mM stock solution of cinnamic aldehyde was added to the appropriate well in column 11 as per the example plate layout.
- One well was left blank.
- All other wells contained 100 µL of the appropriate solvent.
- Serial halving dilutions of the test item were prepared by transferring 100 µL from each dilution into 100 µL of solvent. Pipette tips were discarded after each transfer and then fresh
pipette tips were used to mix each concentration prior to the next transfer.
- The 6.4 mM stock solution of cinnamic aldehyde was diluted from column 11 to column 7 using the same procedure of serial halving dilutions (see table below).

PREPARATION OF THE DILUTION PLATE
- The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 µL of assay medium to each well and then 10 µL solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate.
- Assay medium was 495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.

TREATMENT OF CULTURED PLATES
- 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.
- Assay medium (150 µL) was added to every well of the 96 well plates.
- A 50 µL volume 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.

CELL VIABILITY MEASUREMENT
- After incubation, the transparent plate was removed from the incubator and the plate seal discarded.
- The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium.
- Fresh assay medium (100 µL) was added to each well. MTT solution (10 µL of 5 mg/mL in PBS) was added to each well of the 96-well plate.
- The plate was incubated at 37 ± 2 °C in a humidified atmosphere of 5 % CO2 in air for 4 hours ± 10 minutes.
- The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium.
- DMSO (50 µL) was added to each well. The plate was then placed in the incubator at 37 ± 2 °C, in a humidified atmosphere of 5 % CO2 in air, protected from light, for at least 10 minutes.
- The absorbance value of each well was read using a plate reader with a 540 nm filter.

LUCIFERASE MEASUREMENT
- Luciferase was measured using the Steady Glo Luciferase Assay system kit supplied by Promega (E2550).
- The Steady-Glo luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo buffer to one bottle of Steady-Glo substrate. The reagent was mixed by inversion until the substrate had dissolved.
- Frozen reconstituted reagent was used for tests 1 and 2 and was thawed to room temperature before use.
- After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper.
- Fresh assay medium (100 µL) was added to each well before 100 µL of Steady-Glo luciferase reagent was added to each well of the plate.
- The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed.
- Luminescence (emitted light) was measured using a SpectraMax L luminometer.
- Each plate was read for total photon count with an integration time of 1 second.
- The plates were dark adapted for 1 minute prior to measurement.

NUMBER OF TESTS REQUIRED
- 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.

ASSESSMENT OF RESULTS
- The following parameters were 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) was obtained.
[iii] the IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
- Fold luciferase activity induction was calculated by Equation 1 (see attached document), and the overall maximal fold induction (Imax) was calculated as the average of the individual repetitions.
- EC1.5 was calculated by linear interpolation according to Equation 2 (see attached document), and the overall EC1.5 was calculated as the geometric mean of the individual repetitions.
- Viability was calculated by Equation 3 (see attached document).
- IC50 and IC30 were calculated by linear interpolation according to Equation 4 (see attached document), and the overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.
- For each concentration showing ≥ 1.5 fold luciferase activity induction, statistical significance was calculated (by a two-tailed Student's t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction was statistically significant (p < 0.05). The lowest concentration with ≥ 1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction in cellular viability at the EC1.5 determining concentration. Furthermore, it was checked that no significant cytotoxic effects occur at the lowest concentration leading to ≥1.5 fold luciferase induction.
- The data was visually checked with the help of graphs. Where a statistically non-significant induction above 1.5 fold was observed followed by a higher concentration with a statistically significant induction, results from this test were considered as valid and positive only if the statistically significant induction above the threshold of 1.5 was obtained for a non-cytotoxic concentration.

INTERPRETATION OF RESULTS AND PREDICTION MODEL
- A KeratinoSens prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens prediction is considered negative:
[i] the Imax is ≥1.5 fold and statistically significantly different as compared to the solvent vehicle control (as determined by a two-tailed, unpaired Student's T-test).
[ii] the cellular viability is >70% at the lowest concentration with induction of luciferase activity ≥ 1.5 fold (i.e. at the EC1.5 determining concentration).
[iii] the EC1.5 value is < 1000 µM.
[iv] there is an apparent overall dose-response for luciferase induction (or a biphasic response).
- If in a given test, all of the first three conditions listed above are met, but a clear dose response for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 1000 µM and that do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should also be considered as inconclusive.

TEST ACCEPTANCE CRITERIA
- In order for an assay to be accepted the following criteria must be met:
[i] The luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 (e.g. using a t-test) in at least one of the tested concentrations (4 to 64 µM).
[ii] The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility. In addition, the average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose response of cinnamic aldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
[iii] The average coefficient of variation of the luminescence reading for the negative solvent control (i.e. DMSO) should be below 20% in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results should be discarded.

MAJOR COMPUTERISED SYSTEMS
- Pharmacy test item management: Xybion Pristima
- Plate reader: Wallac EnVision model 2103
- Luminometer: Molecular Devices Spectramax L
- Statistical software: SAS 9.4, SAS Institute Inc. 2002
- All versions of the systems are maintained by Covance.

Positive control results:

- The results for the positive control, cinnamic aldehyde, are shown in Table 2, Table 4, Figure 3 and Figure 4 (attached).
- The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.
- The EC1.5 values of the positive control, cinnamic aldehyde, were 11.13 μM and 3.88 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory (see Table 5, attached).
- The average induction in the three replicates for cinnamic aldehyde at 64 μM were 3.65 and 2.18 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

Run / experiment:

other: Test 1

Parameter:

other: Imax

Value:

3.35

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: > 1.5 fold and statistically significant

Run / experiment:

other: Test 2

Parameter:

other: Imax

Value:

5.98

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: > 1.5 fold and statistically significant

Other effects / acceptance of results:

RESULTS
- The results for the test item are shown in Table 1 (attached) and Table 3 (attached).
- The Imax for the test item was 3.35 in test 1 and 5.98 in test 2. The Imax for both tests was > 1.5 fold and statistically significant compared to the DMSO control.
- The EC1.5 was 5.22 µM and 3.88 µM for tests 1 and 2, respectively.
- The IC30 value was 15.43 µM in test 1 and 47.20 µM in test 2 and the IC50 values were 20.04 µM and 57.74 µM in tests 1 and 2, respectively.
- Figure 1 (attached) and Figure 2 (attached) showed an overall dose-response for luciferase induction.
- The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 15.0 % and 16.8 % for test 1 and 2, respectively, which met the acceptance criterion of below 20 %.

Interpretation of results:

other: substance predicted likely to be a skin sensitiser

Conclusions:

It was concluded that the test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens), supporting the prediction that the test item is likely to be a skin sensitiser.

Executive summary:

GUIDELINE

The test was conducted in accordance with OECD Guideline for the Testing of Chemicals, In Vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation, Guideline 442D, June 2018 and DB-ALM Protocol no 155: KeratinoSens, March 2018.

 

METHODS

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item is likely to be a skin sensitiser using the ARE-Nrf2 Luciferase Test (KeratinoSens).

 

RESULTS

The Imax for the test item was 3.35 in test 1 and 5.98 in test 2. The Imax for both tests was > 1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 5.22µM and 3.88µM for tests 1 and 2, respectively. The IC30 value was 15.43 µM in test 1 and 47.20 µM in test 2 and the IC50 values were 20.04 µM and 57.74 µM in tests 1 and 2, respectively. Graphs showed an overall dose-response for luciferase induction. All acceptance criteria for the positive control, cinnamic aldehyde, were met.

 

CONCLUSION

It was concluded that the test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens), supporting the prediction that the test item is likely to be a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 October 2019 to 25 October 2019

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:

PURPOSE OF THE STUDY
- The DPRA is a chemistry-based assay (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) exploiting the fact that most chemical allergens have electrophilic properties and are therefore able to react with the nucleophilic sidechains of amino acids to form covalent bonds. The underlying rationale of the assay is that if a chemical is capable of reacting with proteins then it has the potential to act as a sensitiser. The endpoint measured in the assay is the percentage depletion over time of two synthetic peptides (containing respectively a cysteine and a lysine amino acid) from the peptide mixtures following an approximate 24-hour (22 to 26 hours) incubation with the test item. The percentage of peptide depletion is calculated by High Performance Liquid Chromatography using ultra-violet detection.
- The DPRA test allows quantification of a chemical’s reactivity and is used to categorize a substance in one of four classes of reactivity to allow discrimination between skin sensitising and non-sensitizing chemicals and thus assesses their sensitisation potential.

PEPTIDE AND POSITIVE CONTROL

SYNTHETIC PEPTIDE CONTAINING CYSTEINE
- Alternative name: Ac-RFAACAA-OH
- Batch number: 1857724
- Stated purity: 96.2 % (by HPLC)
- Molecular weight: 751 g/moL
- Supplier: AnaSpec
- Storage conditions: Frozen, (-10 °C to -30 °C)

SYNTHETIC PEPTIDE CONTAINING LYSINE
- Alternative name: Ac-RFAAKAA-OH
- Batch number: 1857068
- Stated purity: 90.4 % (by HPLC)
- Molecular weight: 777 g/moL
- Supplier: AnaSpec
- Storage conditions: Frozen, (-10 °C to -30 °C)

CINNAMIC ALDEHYDE (POSITIVE CONTROL)
- Batch number: MKCB9907
- Stated purity: 99.1 %
- Molecular weight: 132 g/moL
- Supplier: SAFC
- Storage conditions: Ambient temperature, (15°C to 25°C)
- Expiry/retest date: November 2021

APPARATUS
- High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector.
- Balances fitted with printers: Capability of weighing to 5 decimal places.
- General laboratory apparatus and glassware.

ANALYTICAL REAGENTS
- Propan-2-ol: HPLC grade
- Acetonitrile (ACN): HPLC gradient grade
- Trifluoroacetic acid (TFA): 99 % pure
- Water: Deionised reverse osmosis
- Ammonium Acetate: Analytical reagent
- Sodium Phosphate, monobasic: Analytical reagent
- Sodium Phosphate, dibasic: Analytical reagent
- Ammonium Hydroxide: Analytical reagent
- 100 mM Phosphate buffer, pH 7.5: In house preparation
- 100 mM Ammonium Acetate buffer, pH 10.2: In house preparation
- HPLC Mobile Phase A: 0.1% v/v TFA in Water, in house preparation
- HPLC Mobile Phase B: 0.085% v/v TFA in ACN, in house preparation

ASSESSMENT OF TEST ITEM SOLUBILITY
- The solubility was assessed at a concentration of 100 mM in acetonitrile and propan-2-ol.

PREPARATION OF PEPTIDE STOCK SOLUTIONS
- 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).

PREPARATION OF PEPTIDE CALIBRATION STANDARDS
- Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile.
- The calibration standards 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.

PREPARATION OF STABILITY CONTROLS AND PRECISION CONTROLS
- Stability controls (Reference Control B, n=6 and Reference Control C, n=3) and precision controls (Reference control A) of both peptides were prepared at a concentration of 0.5 mM.
- Reference Control A and Reference Control B were prepared with buffer and acetonitrile.
- Reference Control C was prepared in propan-2-ol.

¬PREPARATION OF POSITIVE CONTROL SOLUTION AND TEST ITEM STOCK SOLUTION
- The positive control chemical (cinnamic aldehyde) was prepared at a concentration of 100 mM in acetonitrile.
- A 100 mM stock solution of the test item was prepared in propan-2-ol.

PREPARATION OF POSITIVE CONTROL AND CYSTEINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS
- Triplicate solutions of test item were diluted with the cysteine peptide to prepare final solutions containing 0.5 mM cysteine and 5 mM of test item.
- Triplicate solutions of the positive control stocks were diluted with the cysteine peptide to prepare final solutions containing 0.5 mM cysteine and 5 mM of positive control.
- For the co-elution control, blank buffer solution was used in place of the cysteine stock solution.

PREPARATION OF POSITIVE CONTROL AND LYSINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS
-- Triplicate solutions of test item were diluted with the lysine peptide to prepare final solutions containing 0.5 mM lysine and 25 mM of test item.
- Triplicate solutions of the positive control stocks were diluted with the lysine peptide to prepare final solutions containing 0.5 mM lysine and 25 mM of positive control.
- For the co-elution control, blank buffer solution was used in place of the lysine stock solution.

INCUBATION
- The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and the vials were then 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.

ANALYSIS
- The concentration of each peptide in the presence of test item and the associated positive controls were quantified by HPLC using UV detection as detailed in the chromatographic section.

INSTRUMENT PARAMETERS
- High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector.
- Column: Agilent Zorbax SB C18, 3.5μm, 100 × 2.1 mm
- Guard column: Phenomenex AJO4286
- Column temperature: 30 °C
- Sample temperature: 25 °C
- Mobile Phase (MP) A: 0.1% TFA in Water
- Mobile Phase (MP) B: 0.085% TFA in CAN
- Gradient: MP A 90 % and MP B 10 % (0 minutes); MP A 75 % and MP B 25 % (20 minutes); MP A 10 % and MP B 90 % (21 minutes); MP A 10 % and MP B 90 % (23 minutes); MP A 90 % and MP B 10 % (23.5 minutes); MP A 90 % and MP B 10 % (30 minutes)
- Flow rate: 0.35 mL/minute
- Stroke volume: 25 μL
- Detector wavelength: UV, 220 nm
- Injection volume: 2 μL (slow draw rate)
- Run time: 30 minutes
- Approximate retention time (cysteine): 11 minutes
- Approximate retention time (lysine): 7 minutes

CALCULATIONS
- The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration.
- The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured.
- Peptide depletion was determined using the equation % Peptide depletion = 100 – (peptide peak area in replicate depletion samples * 100) / mean peptide peak area of reference control samples B or C)

MAJOR COMPUTERISED SYSTEMS
- Chromatography data handling: Waters Empower
- Test substance management: Pristima, Xybion Medical Systems Corporation

Positive control results:

- Historic control data for the positive control are shown in Annex 1 (attached)

Run / experiment:

other: Main Test

Parameter:

other: mean percentage depletion of peptide by test item

Remarks:

cysteine

Value:

3.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

co-elution control

Positive controls validity:

valid

Run / experiment:

other: Main Test

Parameter:

other: mean percentage depletion of peptide by test item

Remarks:

lysine

Value:

-2.43

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

co-elution control

Positive controls validity:

valid

Run / experiment:

other: Main Test

Parameter:

other: overall mean percentage peptide depletion

Remarks:

mean of cysteine and lysine percentage depletion

Value:

1.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

co-elution control

Positive controls validity:

valid

Remarks on result:

other: no or minimal reactivity

Remarks:

negative

Other effects / acceptance of results:

SOLUBILITY ASSESSMENT
- Solubility of the test item in propan-2-ol at a nominal concentration of 100 mM was confirmed.
- The test item was insoluble in acetonitrile at 100 mM.

REACTIVITY ASSESSMENT
- All analytical acceptance criteria for each peptide run were met (see table, attached).
- Mean values are presented in Table 1 (attached).
- Individual values are given in Table 2 (attached).
- Peptide calibration graphs and typical sample chromatograms are presented in Figures 1, 2 and 3 (attached).
- The overall mean peptide depletion was determined to be 1.60 % and equivalent to 'no or minimal reactivity' (see depletion model described in the table, attached). The DPRA prediction for the test material was therefore negative.
- There was co-elution of a peak attributed to the test item with the cysteine peak on the cysteine assay. Based on the co-elution control, the co-eluting peak was equivalent to 7.1 % of the mean cysteine peak and as such is considered not to have impacted on the result of the assay.
- Historic control data for the reference controls are shown in Annex 1 (attached).

Interpretation of results:

other: no or minimal reactivity

Conclusions:

Based on the results of the performed assay, it was not considered likely that the test item is a potential skin sensitiser. This assay is one indicator for skin sensitisation potential and a combination of this study and other in vitro skin sensitisation tests (plus any other data/results available) will give more insight the sensitisation potential of the substance.

Executive summary:

GUIDELINE

The study was conducted in accordance with the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C).

 

METHODS

Solutions of test item were analysed by the validated DPRA analytical method (Covance Analytical Method FIA/M101/15) in both cysteine and lysine containing synthetic peptides.

 

RESULTS

The overall mean peptide depletion was determined to be 1.60 % and equivalent to 'no or minimal reactivity'. The DPRA prediction for the test material was therefore negative.

 

CONCLUSION

Based on the results of the performed assay, it was not considered likely that the test item is a potential skin sensitiser. This assay is one indicator for skin sensitisation potential and a combination of this study and other in vitro skin sensitisation tests (plus any other data/results available) will give more insight the sensitisation potential of the substance.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2019 to 15 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 12-Nousan-8147

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Expert toxicological opinion was that skin sensitisation potential of the test item would be best assessed using the Buehler design.

Species:

guinea pig

Strain:

Hartley

Remarks:

albino

Sex:

male

Details on test animals and environmental conditions:

ANIMALS
- Source: Received from Elm Hill Breeding Labs on 13 November 2019 (preliminary irritation group) and 11 December 2019 (test and naïve control groups)
- Number of animals: 34
- Number of groups: 3
- Animals in preliminary irritation group: 4 young adults
- Animals in test group: 20 young adults (324 to 426 g at experimental start)
- Animals in naïve control group: 10

HUSBANDRY
- Housing: The animals were group housed in suspended stainless steel perforated bottom caging, which conforms to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Enrichment (e.g. tunnel) was placed in each cage and litter paper was changed at least three times per week.
- Animal Room Temperature and Relative Humidity Ranges: 19 to 25 °C and 42 to 62 %. respectively.
- Photoperiod: 12-hour light/dark cycle.
- Acclimation Period: 5 or 26 days.
- Food: PMI Guinea Pig Lab Diet #5025. A designated amount of diet (approximately 20 grams/day) and alfalfa hay cubes (Grainland Select Alfalfa Cubes) were available to each guinea pig.
- Water: Filtered tap water was supplied ad libitum.
- Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water were conducted regularly and the records kept on file at Product Safety Labs.

IDENTIFICATION
- Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
- Animal: Each guinea pig was marked with a colour code and given a sequential animal number assigned to study 51954, which constituted unique identification. Only the sequential animal number was presented in this report.

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.4 mL

Day(s)/duration:

once per week for three weeks (6 hour exposure period)

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.4 mL

Day(s)/duration:

single dose twenty eight days after first induction dose

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

- Preliminary irritation group: 4 young adults
- Test group: 20 young adults
- Naïve control group: 10

Details on study design:

PREPARATION OF THE TEST SUBSTANCE
- Prior to use, the test substance as received and all prepared concentrations were mixed well.
- Solubility testing determined that the test substance was soluble in acetone.

PRELIMINARY IRRITATION TESTING
- A group of animals was used to determine the highest non-irritating concentration (HINIC) of the
test substance prior to the challenge dose.
- The fur was removed by clipping the flanks of each guinea pig. This area was divided into four test sites (two sites on each side of the midline) on each animal.
- The test substance was applied neat (100 %) and also mixed with acetone to yield w/w concentrations of 75 %, 50 % and 25 %.
- Each concentration was applied (0.4 mL each) to a test site using an occlusive 25 mm Hill Top Chamber.
- The sites were wrapped with non-allergenic Durapore adhesive tape.
- After 6 hours of exposure, the chambers were removed and the test sites were gently cleansed with a 3 % soap solution followed by tap water and a clean paper towel to remove any residual test substance.
- Approximately 24 hours after application, each site was evaluated for local reactions (erythema) according to the scoring system described below.
- From these results, the highest non-irritating concentration (HNIC), the highest concentration that produced responses in 4 guinea pigs no more severe than two scores of 0.5 and two scores of zero, was established and used for challenge. The HNIC selected for the challenge phase was 100 %.

PREPARATION AND SELECTION OF ANIMALS
- Within 24 hours prior to each application, the fur of a group of animals was removed by clipping the flanks. After clipping and prior to initiation, the animals were weighed and the skin was checked for any abnormalities.
- Only healthy, naive animals (not previously tested) without pre-existing skin irritation were selected for test. Animals were re-clipped prior to each dose.

INDUCTION PHASE
- Once each week for three weeks, 0.4 mL of the neat test substance was applied to the left side of each test animal using an occlusive 25 mm Hill Top Chamber.
- The chambers were secured in place and wrapped with non-allergenic Durapore adhesive tape to avoid dislocation of the chambers and to minimise loss of the test substance for six hours.
- After the 6-hour exposure period, the chambers were removed and the test sites were gently cleansed with a 3 % soap solution followed by tap water and a clean paper towel to remove any residual test substance.
- Approximately 24 and 48 hours after each induction application, readings were made of local reactions (erythema) according to the scoring system described below.

CHALLENGE PHASE
- Twenty-eight days after the first induction dose, 0.4 mL of the neat test substance (100 %, HNIC) was applied to a naïve site on the right side of each animal as a challenge dose, using the procedures described above. These sites were evaluated for a sensitisation response (erythema) approximately 24 and 48 hours after the challenge application according to the system described below.
- In addition to the test animals, 10 guinea pigs from the same shipment were maintained under identical environmental conditions and were treated with the HNIC of the test substance at challenge only. These animals constituted the “naïve control” group.

SCORING SYSTEM
- No reaction: 0
- Very faint erythema, usually non-confluent (very faint erythema is not considered a positive reaction): 0.5
- Faint erythema, usually confluent: 1
- Moderate erythema: 2
- Severe erythema with or without edema: 3

BODY WEIGHTS
- Individual body weights of the animals were recorded prior to initial induction and again on the day after challenge.

OBSERVATIONS
- All preliminary, test, and control animals were observed for general viability during daily room checks.

STUDY TERMINATION
- Once testing was complete, the animals were released for euthanasia and humanely euthanised.

EVALUATION
- In order to evaluate the sensitization response at challenge, two indices were used: one for incidence and one for severity (Ritz, H. and Buehler, E., 1980) in the test and naïve control animals.
- The incidence index is the ratio of animals with erythema scores greater than 0.5 per number of animals evaluated, and is presented for both the 24 and 48-hour intervals after challenge evaluation as follows:
(i) Incidence Index = Number of erythema scores greater than 0.5 / Number of animals evaluated.
(ii) The severity index is the mean erythema score, and is calculated for both the 24 and 48-hour
intervals after challenge evaluation according to the formula severity index = sum of erythema scores / number of animals evaluated.
- The following criteria were used to classify the test substance as a potential contact sensitizer (Robinson, et al, 1990):
(i) At the 24-hour and/or 48-hour scoring interval, 15 % or more of the test animals exhibit a positive
response (scores > 0.5) in the absence of similar results in the naïve control group.
(ii) The positive reaction at the 24-hour interval must persist to 48 hours in at least one test animal.

CHALLENGE PHASE
- Historical positive control animals (100 % HCA): Eight of ten positive control animals exhibited signs of a sensitization response (faint to moderate erythema [1 to 2) 24 hours after the challenge application. Positive responses persisted at seven positive control sites through 48 hours. Very faint erythema (0.5) was noted at all other sites 24 and/or 48 hours after the challenge application.
- Historical naïve control animals (100 % HCA): Very faint erythema (0.5) was noted at two of five naïve control sites 24 hours after the challenge application, which resolved by 48 hours.

STATISTICAL ANALYSIS
- Statistical analysis was limited to the calculation of the mean severity index scores.

Positive control substance(s):

yes

Remarks:

Hexyl Cinnamic Aldehyde (HCA)

Positive control results:

HISTORICAL POSITIVE CONTROL VALIDATION STUDY
- The procedures used in this study were validated using HCA as a positive control substance.
- The most recent validation and testing (PSL Study #51773) was performed by PSL between 06 November 2019 and 06 December 2019.
- The raw data and report for the study were archived in Product Safety Labs’ Archives Notebook No. 51773: pages 1 to 23. This test was conducted with Hartley strain albino guinea pigs from Elm Hill Breeding Labs following induction and challenge procedures similar to those described above. The results obtained from this testing are presented below.

INDUCTION PHASE
- Historical positive control animals (100 % HCA): Very faint to moderate erytherna (0.5 to 2) was noted at all positive control sites during the induction phase.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

not applicable

Remarks on result:

other: severity zero

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

not applicable

Remarks on result:

other: severity zero

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

not applicable

Remarks on result:

other: severity zero

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

not applicable

Remarks on result:

other: severity zero

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

100 %

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

faint to moderate erythema

Remarks on result:

other: historical positive control validation study

Remarks:

06 November 2019 to 06 December 2019

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

100 %

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

faint to moderate erythema

Remarks on result:

other: historical positive control validation study

Remarks:

06 November 2019 to 06 December 2019

RESULTS

- Preliminary irritation testing scores for the test and historical positive control substances are presented in Table 1 (attached).

- Individual body weights for test, naïve control, and historical positive control animals are presented in Tables 2 and 3, respectively (attached).

- Induction and Challenge Phase skin reaction scores for test, naïve control, and historical positive control animals are presented in Tables 4 through 7, respectively (attached).

- Induction phase test animals (100 % test substance): Very faint to faint erythema (0.5 to 1) was noted at all test sites during the induction phase.

- Challenge phase test animals (100 % test substance): There was no dermal irritation observed at any test site during the challenge phase.

- Naïve control animals (100 % test substance): There was no dermal irritation observed at any naïve control site during the challenge phase.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these findings, and on the evaluation system used, the test item is not considered to be a contact sensitiser. The positive response observed in the historical positive control validation study with HCA validates the test system used in this study.

Executive summary:

GUIDELINE

The study was conducted to determine the potential for the test item to produce sensitisation after repeated topical application. The study was performed in accordance with US EPA Health Effects Test Guidelines, OPPTS 870.2600 (2003), OECD Guidelines for Testing of Chemicals, Test No. 406 (1992), JMAFF 12-Nousan-8147 (2000) and the Official Journal of the European Union, Methods for the Determination of Toxicity and Other Health Effects, Part B.6 (Skin Sensitization), Council Regulation (EC) No. 440/2008.

 

METHOD

The neat test substance was topically applied to twenty healthy test guinea pigs, once each week for a three-week induction period. Twenty-eight days after the first induction dose, a challenge dose of the test substance at its highest non-irritating concentration (1-INIC, determined in the preliminary irritation screen to be 100 %) was applied to a naive site on each guinea pig. A naïve control group (ten animals) was maintained under the same environmental conditions and treated with the test substance at challenge only. Approximately 24 and 48 hours after each induction and challenge dose, the animals were scored for erythema.

 

RESULTS

Very faint to faint erythema (0.5 to 1) was noted at all test sites during the induction phase. There was no dermal irritation observed at any test site during the challenge phase. There was no dermal irritation observed at any naive control site during the challenge phase.

 

CONCLUSION

Based on these findings, and on the evaluation system used, the test item is not considered to be a contact sensitiser. The positive response observed in the historical positive control validation study with HCA validates the test system used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro

 

ARE-Nrf2 Luciferase Test (KeratinoSens)

A key study was conducted in accordance with OECD Guideline for the Testing of Chemicals, In Vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation, Guideline 442D, June 2018 and DB-ALM Protocol no 155: KeratinoSens, March 2018.

 

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item is likely to be a skin sensitiser using the ARE-Nrf2 Luciferase Test (KeratinoSens).

The Imax for the test item was 3.35 in test 1 and 5.98 in test 2. The Imax for both tests was > 1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 5.22 µM and 3.88 µM for tests 1 and 2, respectively. The IC30 value was 15.43 µM in test 1 and 47.20 µM in test 2 and the IC50 values were 20.04 µM and 57.74 µM in tests 1 and 2, respectively. Graphs showed an overall dose-response for luciferase induction. All acceptance criteria for the positive control, cinnamic aldehyde, were met.

 

It was concluded that the test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens), supporting the prediction that the test item is likely to be a skin sensitiser.

 

Direct Peptide Reactivity Assay (DPRA)

A key study was conducted in accordance with the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C).

 

Solutions of test item were analysed by the validated DPRA analytical method (Covance Analytical Method FIA/M101/15) in both cysteine and lysine containing synthetic peptides.

 

The overall mean peptide depletion was determined to be 1.60% and equivalent to 'no or minimal reactivity'. The DPRA prediction for the test material was therefore negative.

 

Based on the results of the performed assay, it was not considered likely that the test item is a potential skin sensitiser. This assay is one indicator for skin sensitisation potential and a combination of this study and other in vitro skin sensitisation tests (plus any other data/results available) will give more insight the sensitisation potential of the substance.

In vivo

Buehler study 

The key study was conducted using the Buehler method to determine the potential for the test item to produce sensitisation after repeated topical application. The study was performed in accordance with US EPA Health Effects Test Guidelines, OPPTS 870.2600 (2003), OECD Guidelines for Testing of Chemicals, Test No. 406 (1992), JMAFF 12-Nousan-8147 (2000) and the Official Journal of the European Union, Methods for the Determination of Toxicity and Other Health Effects, Part B.6 (Skin Sensitization), Council Regulation (EC) No. 440/2008.

 

The neat test substance was topically applied to twenty healthy test guinea pigs, once each week for a three-week induction period. Twenty-eight days after the first induction dose, a challenge dose of the test substance at its highest non-irritating concentration (1-INIC, determined in the preliminary irritation screen to be 100 %) was applied to a naive site on each guinea pig. A naïve control group (ten animals) was maintained under the same environmental conditions and treated with the test substance at challenge only. Approximately 24 and 48 hours after each induction and challenge dose, the animals were scored for erythema.

 

Very faint to faint erythema (0.5 to 1) was noted at all test sites during the induction phase. There was no dermal irritation observed at any test site during the challenge phase. There was no dermal irritation observed at any naive control site during the challenge phase.

 

Based on these findings, and on the evaluation system used, the test item is not considered to be a contact sensitiser. The positive response observed in the historical positive control validation study with HCA validates the test system used in this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Available data from validated in vitro tests supplemented by results of investigation in vivo (Buehler method) indicate that classification in accordance withEU Regulation (EC) No. 1272/2008 is not required.