[No public or meaningful name is available]

Endpoint summary

Description of key information

SKIN SENSITISATION: Skin sensitiser, Cat 1B

Skin sensitisation of the registered substance has been assessed using a weight of evidence approach combined with a read-across to structural analog substance. The outcome is based on the DPRA (OECD TG 442C) and KeratinoSens (OECD TG 442D) tests conducted with both target and source substances, and the in vivo LLNA study (OECD TG 429) on the source substance. Similar effects in in vitro studies indicate the similar skin sensitising properties between the target and source substances and gives confidence to use the in vivo potency results from the source substance.

Key Event 1. OECD TG 442C (DPRA)

Target substance: negative (2019). 1.41% +-0.71% SPCC depletion. 2.18% +-2.93% SPCL depletion.

Source substance: negative (2020). 3.24% +-1.27% SPCC depletion. 0.19% +-0.29% SPCL depletion.

Neither precipitation nor phase separation were assessed during the two OECD 442C studies. However in such circumstances, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. 

Key Event 2: OECD TG 442D (KeratinoSens)

Target substance: positive (2019). EC1.5 9.24 / 12.61 μM, IC50 47.56 / 105.44 μM  (tests 1 / 2)

Source substance: positive (2019). EC1.5 13.83 / 9.38 µM, IC50 97.79 / 140.82 µM (tests 1 / 2)

 

In vivo study: OECD TG 429 (LLNA)

Source substance:  positive, EC3=9% (2013)

 

RESPIRATORY SENSITISATION: no data available

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08-08-2019 to 10-10-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative 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).
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 prepared as well.

- Preparation of the test chemical solutions:
100 mM stock solutions of the test item were prepared in acetonitrile for both analytical occasions.
Triplicate solutions each of test item and the positive control stocks were diluted with the Cysteine peptide to prepare final solutions containing 0.5 mM Cysteine and 5 mM of either the test item or the positive control. Also triplicate solutions each of test item and the positive control stocks were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either the test item or the positive control.

- Preparation of the positive controls, reference controls and co-elution controls:
Positive controls: The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile.
Triplicate solutions each of test item and the positive control stocks were diluted with the Cysteine peptide to prepare final solutions containing 0.5 mM Cysteine and 5 mM of either the test item or the positive control. Also triplicate solutions each of test item and the positive control stocks were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either the test item or the positive control.

Reference controls: Stability controls (Reference Control B, n=6) 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.

Co-elution controls: For the co-elution controls, blank buffer solutions were used in place of the Cysteine and Lysine stock solutions.

INCUBATION
- Incubation conditions: 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 injection on the analysis run. Prior to injection of the samples the appearance of the samples in the vials was assessed and documented again.

- Precipitation noted: Precipitation not observed.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys:
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 prepared as well.
Calibration curve is available in the full study report. The calibration curve met the acceptability criteria: standard calibration curve having an r2 > 0.99.

- Verification of the suitability of the HPLC for test chemical and control substances:
The mean peptide concentration of Reference controls B was 0.491 mM and 0.507 mM for both Cysteine and Lysine. they both were within the acceptance criteria of 0.45-0.55 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN, acetonitrile) used to dissolve the test item did not impact the Percent SPCC nor SPCL Depletion.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: The relative peptide concentration is measured by high-performance liquid chromatograph (HPLC) with gradient elution and spectrophotometric detection at 220 nm.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

1.41 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

2.18 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No phase separation neither precipitation occurred after the incubation period for both Cysteine and Lysine samples.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C.

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 reference controls A to C: All criteria met.
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): 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.

Table 1: Acceptability of the DPRA

	Peptide	Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r^2>0.99	60.8-100 (SD <14.9%)	0.45-0.55 mM (CV <14.9%)	SD < 14.9%
	Lysine	r^2>0.99	40.2-69.0 (SD <11.6%)	0.45-0.55 mM (CV <14.9%)	SD < 11.6%
Achieved results	Cysteine	r^2>0.999	71.5 (SD 0.21%, n=3)	B: 0.491 mM (CV=0.56%, n=6)	SD 0.71%
	Lysine	r^2>0.999	61.0 (SD 2.76%, n=3)	B: 0.507 mM (CV=1.68%, n=6)	SD 2.93%

CV: Coefficient of Variation

Table 2: Depletion of peptide in the presence of the test item

	Mean peak area of reference control (µV.sec)	Mean peak area of peptide with test item (µV.sec)	Mean depletion (%)
Cysteine	B: 903730 (n=6)	890990 (n=3)	1.41
Lysine	B: 721990 (n=6)	706290 (n=3)	2.18

Table 3: Results of the DPRA with the test item

	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test Item	1.41%	0.71%	2.18%	2.93%	1.79%	Negative: No or minimal reactivity

SD: Standard Deviation

Interpretation of results:

GHS criteria not met

Remarks:

Test item was negative in the DPRA and was classified in the "no or minimal reactivity class when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Conclusions:

The test item gave a negative in DPRA and was classified in the "Negative: No or 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.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C, 18 June 2019) was to assess the reactivity and sensitising potential of the test item, ST 08 C 19.  Solutions of this test item were incubated with two synthetic peptides (containing respectively a cysteine and a lysine amino acid) for approximate 24 hours and then the concentration of each peptide was measured relative to a series of peptide control solutions.

Solutions of the test item in acetonitrile were successfully analysed by the validated DPRA analytical method (analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides.  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. 

 

Under the conditions of this study, there was no or minimal depletion (mean depletion of 1.79%) of either the Cysteine or Lysine peptides in the presence of the test item, the DPRA assay categorises this test item as negative and hence it is predicted not to be a skin sensitiser. 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27-08-2019 until 02-01-2020

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 KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution:
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 chemical serial dilutions:
In the main experiment the test item was dissolved in DMSO at 200 mM (clear colourless solution). 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. 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 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.

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. Test concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.98 (final concentration DMSO of 1%) in the first and second experiment.
All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

- Preparation of the positive controls:
The positive control used is Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020). Prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO. 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. 200μL of the 6.4 mM stock solution of cinnamic aldehyde was added to the appropriate well in column 11. 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 as with the test item.

- Preparation of the solvent, vehicle and negative controls:
The vehicle control was 1% DMSO in exposure medium.

DOSE RANGE FINDING ASSAY:
- Highest concentration used:
200 mM was selected as highest concentration for the main assay (highest dose required in the current guideline).

- Solubility in solvents:
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.

- Solubility in incubation medium:
Not reported.

- Cytotoxicity assessment performed ????
- Final concentration range selected on basis of: [describe] ????

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3 for each test item concentration.

- Number of repetitions: 2 independent experiments.

- Test chemical concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.98 μM (final concentration DMSO of 1%) in the first and second experiment.

- Application procedure: 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.

- Exposure time: 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.

- Study evaluation and decision criteria used: The following parameters were calculated in the KeratinoSens™ test method:
- the maximal average fold induction of luciferase activity (Imax) value observed at any
concentration of the tested chemical and positive control;
- 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; and
- the IC50 and IC30 concentration values for 50% and 30% reduction of cellular
viability.
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:
- 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);
- 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);
- the EC1.5 value is < 1000 μM / < 200 μg/mL;
- 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 / < 200 μg/mL and that do not reach
cytotoxicity (< 70% viability) at the maximal tested concentration should also be considered
as inconclusive.

- Description on study acceptance criteria:
In order for an assay to be accepted the following criteria must be met:
- 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).
- 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.
- 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.


SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): Passage 16 of the cells only reached 60% confluence, however, as the cells were actively growing and there were sufficient cells to perform the assay, there was no impact on the study.

- Incubation conditions: The plates were incubated at at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air.

- Washing conditions: The cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution.

- Precipitation noted: not reported.


LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: Molecular Devices Spectramax L.

- Plate used: 96 wells.

- Lysate preparation: Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). 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. 100 μL of fresh assay
medium 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.


DATA EVALUATION
- Cytotoxicity assessment: 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. 100 μL fresh assay
medium was added to each well. 10 μL of MTT solution (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. 50 μL of DMSO 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.

- Prediction model used: According to Figure 1 of the OECD 442D guideline (2018).

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

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 < 4.00 μM and 12.27 μM
for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 μM were 4.38 and 2.57 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

12.61 µM

Cell viability:

>70%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

9.24 µM

Cell viability:

>70%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Imax value = 3.62

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Imax value= 3.63

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Value:

105.44 µM

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Value:

47.56 µM

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

87.63 µM

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

41.9 µM

Outcome of the prediction model:

positive [in vitro/in chemico]

Table 1: Overview Luminiscence Induction and Cell Viability of Test Item in Experiment 1 and 2

Concentration (μM)	0,98	1,95	3,91	7,81	15,63	31,25	62,5	125	250	500	1000	2000
Luminiscence - Exp1	0,81	0,91	1,09	1,42	1,88*	2,03*	2,85*	3,62*	-0,18	-0,19	-0,08	0,01
Viability (%) - Exp1	110,26	111,76	104,62	92,37	118,23	107,63	-2,78	0,3	-0,83	0,08	0,08	0,3
Luminiscence - Exp2	0,84	0,9	0,97	1,31	1,62	2,07	2,77	3,63	0	0	0	0
Viability (%) - Exp2	93,28	104,77	97,74	100,02	111,21	118,53	98,23	28,02	8,12	6,24	6,34	6,14

*p<0.05 Student's test

 

Table 2: Overview Luminiscence Induction and Cell Viability Positive Control cinnamic aldehyde in Experiment 1 and 2

Concentration (μM)	4	8	16	32	64
Luminiscence - Exp1	1,74*	1,78*	2,06*	2,49*	4,38*
Viability (%) - Exp1	106,73	91,92	90,04	17,59	101,09
Luminiscence - Exp2	1,37	1,44	1,55*	1,91*	2,57*
Viability (%) - Exp2	155,08	152,8	139,92	122,99	120,42

*p<0.05 Student's test

 

Table 3: Overview EC_1.5, I_max, IC₃₀ and IC₅₀ values of Experiment 1 and 2 

	EC1.5	Imax	IC30 (μM)	IC50 (μM)
Test item Exp1	9,24	3,62	41,9	47,56
Test item Exp2	12,61	3,63	87,63	105,44
Positive Control Exp1	<4,00	4,38	N/A	N/A
Positive Control Exp2	12,27	2,57	N/A	N/A

N/A: Not applicable. 

Interpretation of results:

other: Deviations from study plan: Incubation time of DMSO for measuring cell viability Section 5.6 of the study plan states that after incubation with MTT that DMSO will be added to each well of the plate and incubated for at least 10 minutes at 37 ± 2°C. In th

Remarks:

Incubation time of DMSO for measuring cell viability -- Section 5.6 of the study plan states that after incubation with MTT that DMSO will be added to each well of the plate and incubated for at least 10 minutes at 37 ± 2°C. In the raw data for the test performed between 09 September 2019 and 12 September 2019 the incubation with DMSO end time has been omitted, however, the time between the start of incubation and the plate read was 16 minutes therefore it can be concluded that the plate was incubated for at least 10 minutes. In addition, the control wells gave the expected response. The raw data for the test performed between 23 September 2019 and 26 September 2019 states that the plate was incubated with DMSO for 3 minutes, however, the time between the start of incubation and the plate read was 15 minutes therefore it can be concluded that the plate was incubated longer than the 3 minutes stated. In addition, the control wells gave the expected response. There was no impact on the scientific integrity of the study. Cell Confluency -- Section 5.1.2 of the study plan states that the cells will be subcultured when they have reached 80-90% confluence. Passage 16 of the cells only reached 60% confluence, however, as the cells were actively growing and there were sufficient cells to perform the assay, there was no impact on the study.

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 considered to be a skin sensitizer.

Executive summary:

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 Imax for ST 08 C 19 was 3.62 in test 1 and 3.63 in test 2. The Imax for both tests was > 1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 9.24 μM and 12.61 μM for tests 1 and 2, respectively. The IC30 value was 41.90 μM in test 1 and 87.63 μM in test 2 and the IC50 values were 47.56 μM and 105.44 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 considered to be a skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar
toxicological properties because they have common structural features in the same relatives positions. Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable chemical similarity. Impurities present in the target substance are configurational isomers of the main constituents and are therefore not impacting the chemical similarity between the source and target substances. Further information is included in attachment to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
The source and the target have similar physico-chemical, toxicological properties and because of common metabolism they share common or have similar breakdown products and therefore potential mechanisms of action.
The available information on composition, physico-chemistry and toxicity suggests that the substances (source and target) share sufficient common properties, so that read across may be undertaken for the skin sensitisation endpoint. Further information is included in attachment to IUCLID section 13.

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0.186 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

3.24 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Interpretation of results:

other: The results obtained in this DPRA will be considered within a weight of evidence assessment for C&L purposes

Conclusions:

The results obtained in this DPRA on an analog substance will be considered within a weight of evidence assessment

Executive summary:

The study was performed on a source substance in order to assess the reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) (in chemico Direct peptide reactivity assay (DPRA)) according to the testing guideline OECD 442 C (2019) and under GLP.

 

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 photodiode array (PDA) detection at 220 nm and 258 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 (ACN) was found to be an appropriate solvent to dissolve the test item. 

All acceptability criteria were met according to the requirements of the OECD 442 C guideline. 

In the cysteine reactivity assay the test item showed 3.24% SPCC depletion while in the lysine reactivity assay the test item showed 0.186% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.71%. As a result the DPRA assay categorises the source substance as negative.

The overall skin sensitisation profile of the target substance will be considered within a weight of evidence approach in order to conclude on its skin sensitisation potential.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar
toxicological properties because they have common structural features in the same relatives positions. Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable chemical similarity. Impurities present in the target substance are configurational isomers of the main constituents and are therefore not impacting the chemical similarity between the source and target substances. Further information is included in attachment to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
The source and the target have similar physico-chemical, toxicological properties and because of common metabolism they share common or have similar breakdown products and therefore potential mechanisms of action.
The available information on composition, physico-chemistry and toxicity suggests that the substances (source and target) share sufficient common properties, so that read across may be undertaken for the skin sensitisation endpoint. Further information is included in attachment to IUCLID section 13.

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

9.38 µM

Cell viability:

>70%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

13.83 µM

Cell viability:

>70%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Value:

140.82 µM

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Value:

97.79 µM

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

105.76 µM

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

84.38 µM

Interpretation of results:

other: The test conducted on an analog substance gave 2 out of 2 positive experiments (each in triplicate). The result will be considered within a weight of evidence assessment for C&L purposes

Conclusions:

Under the condition of the study, the analog substance ST 12 C 19 is considered as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) since >1.5-fold induction was observed at test concentrations of ≤1000 µM with a cell viability of >70% compared to the vehicle control.

Executive summary:

The source substance, structural analog to the registered substance, was tested to evaluate its ability to activate the  antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay according to the OECD 442D (2018) guideline.

The test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM (stock solution) and then further diluted to obtain the following tested concentrations range: 0.98 – 2000 µM (2-fold dilution series). Cinnamic aldehyde was used as positive control at the final concentration range 4 - 64 µM in 1% DMSO. Two independent experiments (with replicate points for each concentration) were performed. Both experiments passed the acceptance criteria.

Thesource substance showed toxicity (IC30 values of 84.38 µM and 105.76 µM and IC50 values of 97.79 µM and 140.82 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 13.83 µM and 9.38 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 3.18-fold and 10.05-fold in experiment 1 and 2 respectively.

Therefore, the source substance, structural analog of the registered substance is considered as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

 

The overall skin sensitisation profile of the target substance will be considered within a weight of evidence approach in order to conclude on its skin sensitisation potential.

 

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar
toxicological properties because they have common structural features in the same relatives positions. Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable chemical similarity. Impurities present in the target substance are configurational isomers of the main constituents and are therefore not impacting the chemical similarity between the source and target substances. Further information is included in attachment to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
The source and the target have similar physico-chemical, toxicological properties and because of common metabolism they share common or have similar breakdown products and therefore potential mechanisms of action.
The available information on composition, physico-chemistry and toxicity suggests that the substances (source and target) share sufficient common properties, so that read across may be undertaken for the skin sensitisation endpoint. Further information is included in attachment to IUCLID section 13.

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Not applicable

Key result

Parameter:

SI

Remarks:

additional test

Value:

2.12

Test group / Remarks:

5% v/v in acetone/olive oil 4:1

Remarks on result:

other: negative

Key result

Parameter:

SI

Value:

6.19

Test group / Remarks:

25% v/v in acetone/olive oil 4:1

Remarks on result:

other: positive

Key result

Parameter:

SI

Value:

13.93

Test group / Remarks:

50% v/v in acetone/olive oil 4:1

Remarks on result:

other: positive

Key result

Parameter:

SI

Value:

15.06

Test group / Remarks:

100%

Remarks on result:

other: positive

Key result

Parameter:

EC3

Value:

9

Test group / Remarks:

25% v/v in acetone/olive oil 4:1

Remarks on result:

other: positive

Interpretation of results:

other: the results of LLNA performed on a source substance showed positive results. The result will be considered within a weight of evidence assessment for C&L purposes

Conclusions:

Under the test conditions, the source substance is a contact sensitiser (Category 1B) according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

A study was performed to assess the skin sensitisation potential of a source substance, structural analog to the registered substance in the CBA/Ca (CBA/CaOlaHsd) strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a test item concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 μl (25 μl per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. A further group of five animals was treated with acetone/olive oil 4:1 alone. In order to determine the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value), and at the request of the Study Sponsor, an additional group of five animals was treated with the test item at a concentration of 5% v/v in acetone/olive oil 4:1. An additional group of five animals was treated with acetone/olive oil 4:1 alone.

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No visual local skin irritation or excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted at any dose concentration evaluated. Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

 

The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 2.12, 6.19, 13.93 and 15.06 at 5, 25 and 50% v/v in acetone/olive oil 4:1 and 100%, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 9%.

 

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 6.29, when tested at 25 % v/v. The test system was therefore considered to be valid.

 

Under the test conditions, the source substance is a contact sensitiser (Category 1B) according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.

The overall skin sensitisation profile of the target substance will be considered within a weight of evidence approach in order to conclude on its skin sensitisation potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The registered substance is assigned to GHS Classification: Skin Sensitisation Category 1B. Further information concerning the weight of evidence conclusion is attached by the applicant to this endpoint.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on a weight of evidence approach, the registered substance is classified as Skin sensitiser Cat 1 B according to CLP Regulation 1272/2008 (EC) and to GHS.