2-methylpentyl 2-hydroxybenzoate

Endpoint summary

Description of key information

Weight of Evidence: skin sensitising, 2021

1. Molecular initiating Key Event 1: positive, DPRA, OECD TG 442C, 2020

Cysteine reactivity assay : (mean ; n=3): 1.1% ± 1.2% ; negative : No or minimal reactivity : noting some phase separation was observed within the cysteine assay

Lysine reactivity assay : (mean ; n=3): 21.4% ± 23.0% ; positive : possible moderate reactivity : noting some phase separation was observed within the Lysine assay ; not all replicate variability criteria were met.

Assessment indicates using the Cysteine 1:10 / Lysine 1:50 prediction model :

(i) the estimated Mean of SPCC and SPCL depletion was 11.3% and in the > 6.38 < 22.62 - predicted low reactivity class

(ii) the estimated result for lysine is suggested as possible: > 22.62 < 42.47 - moderate reactivity class

The result will be considered within a weight of evidence for assessment for purposes

2. Molecular initiating Key Event 2: negative (no LC induction > 1.5 observed in 2 experiments and up to 2000 μM) (n=3 replicates), KeratinoSens, OECD TG 442D, 2020

3(a). Molecular initiating Key Event 3: positive (mean SMV Decision Value (DV) of +2.02, and since SMV DV> 0 (n ≥ 2) predicted to be skin sensitising), GARDSkin Assay, 2021

3(b). POTENCY: weak skin sensitiser (GHS Skin Sensitiser 1B) : median SMV Decision Value (DV) of -0.915, and since median SMV DV < 0 (n ≥ 2) = weak skin sensitising, GARDpotency Assay, 2021

4. Expert Judgement: In accordance with REACH Regulation (EC) No. 1907/2006 Annex XI, section 1.2 – weight of evidence, justification for not performing further skin sensitisation in vivo studies includes expert assessment: The category: salicylates, were reviewed by Belsito et al., (2007). A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients. Food and Chemical Toxicology, 45, S318–S361. Therein the salicylates, with the exception of benzyl salicylate, were considered based on all the available data to have ‘some potential for skin sensitization’ but that in general they possess either ‘no or very limited skin sensitization potential’ in humans. As such the conclusion was that for the category in general: it could be inferred that members therein were either non-classified or GHS Skin Sensitisation category 1B (i.e. low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed). This correlates with the conclusion of the GARDpotency (2021) assay, conducted on the substance using a method consistent with OECD Test Guideline Program (TGP no. 4.106) and/or EURL ECVAM test method: TM2011-09 (EU). Additional references on the test method are provided in R. Gradin et al. (2020). The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432. The available data indicates a weight of evidence that the substance is a skin sensitiser and/or should be classified within GHS Skin Sensitisation category 1B. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.3, July 2017) further testing is not scientifically justified.

 

Conclusion: Within the battery of in chemico and in vitro test assays, there are equivocal to positive predictions for sensitisation. The test item appears to be either a non-sensitiser to weak sensitiser. Using a precautionary principle conclusion through evaluation of all the available information, the substance is considered a weak sensitiser and to have a low potency (e.g. EC3 >> 2%) based on the weight of evidence. (GHS Classification: Skin Sensitisation Category 1B).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05-03-2020 to 19-03-2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. Relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017). The result will be considered within a weight of evidence assessment for C&L purposes.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

All acceptance criteria were met, with the exception of the SD value of the SPCL depletion which was above the acceptability criteria for the DPRA. See 'other effects/acceptance of results' and 'Applicant's summary and conclusions' for more information.

GLP compliance:

yes

Remarks:

Guideline study performed under GLP. Relevant validity criteria were met with acceptable restrictions. Test method considered to cover Key Event-1 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- HPLC-PDA (UV) methodology are reported in the full study report.

PREPARATION OF TEST SOLUTIONS
- Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol and ethanol. At a concentration of 100 mM, the test item was soluble in ACN. Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in the study. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The dissolution of the test item in the SPCC and SPCL assay buffers was also evaluated by diluting the test item stock solution in the buffer-based incubation mixtures. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of the peptide/derivative stock solutions: [Synthetic Peptide Containing Cysteine (SPCC) and Synthetic Peptide Containing Lysine (SPCL)]
1. Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.0 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
2. Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.2 mg of SPCL in 19.69 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes (and/or sonication if necessary).
- Preparation of the test chemical solutions: For both the cysteine and lysine reactivity assay 45.2 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 2.03 mL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the a clear solution being formed was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
- Preparation of the positive controls, reference controls and co-elution controls:
1. Cysteine: SPCC Reference Control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN. The SPCC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The mean peptide concentration of Reference Controls A was 0.516 ± 0.002 mM while the mean peptide concentration of Reference Controls C was 0.507 ± 0.002 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
2. Lysine: SPCL Reference Control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN. The SPLC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report). The mean peptide concentration of Reference Controls A was
0.482 ± 0.011 mM while the mean peptide concentration of Reference Controls C was 0.496 ± 0.009 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
3. Positive controls: PC samples were prepared from 750 μL ’stock solution’ of 0.667 mM SPCC or SPCL (as applicable), 250 μL Cinnamic aldehyde solution (100 mM in ACN) and (in case of Cysteine) further 200 μL ACN.

INCUBATION
- Incubation conditions: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC analysis the samples were visually inspected for precipitation. Samples that showed phase separation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.
- Precipitation noted: (i) In the Cysteine reactivity assay : upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the co-elution control (CC) and the test item samples ; (ii) In the Lysine reactivity assay: upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the co-elution control (CC) and the test item samples.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Yes. HPLC-PDA (UV) methodology are reported in the full study report. The concentration of SPCC or SPCL was spectrophotometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards. The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C using standard formula (see: OECD TG 442C).
- Verification of the suitability of the HPLC for test chemical and control substances: In addition to the above, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA EVALUATION
- Cys and Lys peptide detection wavelength: See above. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm for both the Cysteine Reactivity Assay and Lysine Reactivity Assay, are presented in the full study report.

- ACCEPTABILITY CRITERIA:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.9994 and SPLC r2 = 1.0000)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 72.8% ± 0.2% and SPCL 65.0% ± 0.9%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.2% and SPCL PC : SD = 0.9%)
(iv) mean peptide concentration of Reference Controls A, C is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C reference controls: 0.516 ± 0.05 mM, and 0.507 ± 0.002 mM ; Lysine A, C reference controls: 0.482 ± 0.011 mM and 0.496 ± 0.009 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 1.3% ; Lysine: Reference Controls B and C was 2.9%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm and 258 nm was observed at the retention time of SPCC and SPLL. For the test item/A-cys samples, the mean SPCC A220/A258 area ratio was 37.88. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 30.69. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met.
- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=750.9) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=775.9) – full details on source provided in full study report.
- Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; 99.1%) – full details on source provided in full study report.
Negative control (NC): Vehicle = Acetonitrile (ACN)
Evaluation of results: In accordance with OECD TG 442C – Table 1.
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity (Positive)
> 22.62 < 42.47 moderate reactivity (Positive)
> 6.38 < 22.62 low reactivity (Positive)
< 6.38 minimal reactivity (Negative)

Positive control results:

- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 72.8% ± 0.2% (high reactivity)
- PC LYS-peptide depletion (mean): 65.0% ± 0.9% (high reactivity)

Key result

Run / experiment:

mean

Parameter:

mean cystein depletion

Remarks:

Mean (%) Cys-peptide depletion

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: (mean ; n=3):1.1% ± 1.2% ; negative : No or minimal reactivity

Remarks:

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

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Remarks:

Mean (%) Lys-peptide depletion

Value:

21.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: (mean ; n=2): 24.4% ± 23.0% ; positive ; moderate reactivity

Remarks:

n = 2 ; one sample excluded. See 'any other information on results incl. tables' for further information

Outcome of the prediction model:

other: See comments provided below in 'Any other information on results incl. tables' and/or 'Applicant's summary and conclusion'

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.
- Other:
(i) In the Cysteine reactivity assay : upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the co-elution control (CC) and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.
(ii) In the Lysine reactivity assay: upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the co-elution control (CC) and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met , with the exception of the mean Percent SPCL Depletion variability, see comments below.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.9994 and SPLC r2 = 1.0000)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 72.8% ± 0.2% and SPCL 65.0% ± 0.9%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 0.2% and SPCL PC : SD = 0.9%)
(iv) mean peptide concentration of Reference Controls A, C is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C reference controls: 0.516 ± 0.05 mM, and 0.507 ± 0.002 mM ; Lysine A, C reference controls: 0.482 ± 0.011 mM and 0.496 ± 0.009 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 1.3% ; Lysine: Reference Controls B and C was 2.9%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm and 258 nm was observed at the retention time of SPCC and SPLL. For the test item/A-cys samples, the mean SPCC A220/A258 area ratio was 37.88. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 30.69. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met with the exception of: the mean Percent SPCL Depletion for the test item was 21.4% ± 23.0% (estimated value). The standard deviation value of the SPCL depletion was above the acceptability criterium for the DPRA as stated in the OECD 442C guideline. As this was caused by the occurrence of a phase separation in the test item samples in combination with interference of a test item related peak with SPCL, DPRA results were accepted.

Table 1.0 – Acceptability of the DPRA

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r2) standard calibration curve	>0.99	0.9994	>0.99	1.0000
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.516 ± 0.002	0.50 ± 0.05	0.482 ± 0.011
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.507 ± 0.002	0.50 ± 0.05	0.496 ± 0.009
CV (%) for RC samples B and C	<15.0	1.3	<15.0	2.9
Mean peptide depletion PC (cinnamic aldehyde) (%)	60.8-100	72.8	40.2-69.0	65.0
SD of peptide depletion PC (cinnamic aldehyde) (%)	<14.9	0.2	<11.6	0.9
SD of peptide depletion for the test item (%)	<14.9	1.2	<11.6	23.0 #

Where: RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation

 

Table 2.0 – Results of the DPRA with the test item

	SPCC depletion (CYSTEINE)		SPCL depletion (LYSINE)		Mean of SPCC and SPCL depletion
	Mean	± SD	Mean	± SD
Test item	1.1%	±1.2%	21.4% #1	±23.0%	11.3%

#1 : based on only two replicates.

 

Note:

Within the results of the lysine assay:

(1) In the CC sample no peak was observed at the retention time of SPCL. This indicated that there was no co-elution of the test item with SPCL. For the test item/A-lys samples, the mean SPCL A220/A258 area ratio was 29.42. Since this was within the 27.62-33.76 range, this again indicated that there was no co-elution of the test item with SPCL, however, in the test item/A-lys-2 and the test item/A-lys-3 samples it appeared that a test item related peak interfered with the SPCL peak at both wavelengths.

(2) As the overlap in retention time between the test item related peak and SPCL at the 220 nm wavelength was incomplete, an estimation of the Percent SPCL Depletion was made. For this estimation, the data of the test item/A-lys-3 sample was excluded from calculations as the SPCL peak area (1767491) was much higher than that of the mean of the peptide areas for the nine Reference Controls B and C (1581310). Therefore the Lysine reactivity (positive) prediction was based on only two replicates.

 

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed in the test item samples. An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion were presented. In the cysteine reactivity assay the test item showed 1.1% SPCC depletion while in the lysine reactivity assay the test item showed 21.4% SPCL depletion (estimated value). The mean of the SPCC and SPCL depletion was 11.3% (estimated value) and as a result the test item was considered to be positive in the DPRA. As the overlap in retention time between the test item and SPCL was incomplete, the SPCL depletion was estimated and used in the Cysteine 1:10 / Lysine 1:50 prediction model, however, assignment to a reactivity class was not (formally) made (within the study).

 

Conclusion:

The test item gave a positive in the DPRA and was not classified into a reactivity class (formally, within the study) due phase separation being observed in the incubation period in SPCC and SPCL test item-samples.

However applicant assessment indicates using the Cysteine 1:10 / Lysine 1:50 prediction model :

(i) the Mean of SPCC and SPCL depletion was in the > 6.38 < 22.62 - predicted low reactivity (Positive) class

(ii) the estimated result for lysine is suggested as possible: > 22.62 < 42.47 - predicted moderate reactivity (Positive) class

The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Interpretation of results:

other: The test item gave a positive in the DPRA and was not classified into a reactivity class (formally, within the study) due phase separation being observed. The result will be considered within a weight of evidence assessment for C&L purposes

Conclusions:

The test item gave a positive in the DPRA and was not classified into a reactivity class (formally, within the study) due phase separation being observed in the incubation period in SPCC and SPCL test item-samples.
However applicant assessment indicates using the Cysteine 1:10 / Lysine 1:50 prediction model :
(i) the Mean of SPCC and SPCL depletion was in the > 6.38 < 22.62 - predicted low reactivity (Positive) class
(ii) the estimated result for lysine is suggested as possible: > 22.62 < 42.47 - predicted moderate reactivity (Positive) class
The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Executive summary:

The study was performed to the OECD TG 442C in chemico Direct Peptide Reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and 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. The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the SPCC depletion for the test item, were all within the acceptability criteria for the DPRA. the standard deviation value of the SPCL depletion was above the acceptability criterium for the DPRA as stated in the OECD 442C guideline. As this was caused by the occurrence of a phase separation in the test item samples in combination with interference of a test item related peak with SPCL, DPRA results were accepted. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed in the test item samples. An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion were presented. In the cysteine reactivity assay the test item showed 1.1% SPCC depletion while in the lysine reactivity assay the test item showed 21.4% SPCL depletion (estimated value). The mean of the SPCC and SPCL depletion was 11.3% (estimated value) and as a result the test item was considered to be positive in the DPRA. As the overlap in retention time between the test item and SPCL was incomplete, the SPCL depletion was estimated and used in the Cysteine 1:10 / Lysine 1:50 prediction model, however, assignment to a reactivity class was not made.

 

Applicant assessment indicates: the test item gave a positive in the DPRA and was not classified into a reactivity class (formally, within the study) due phase separation being observed in the incubation period in SPCC and SPCL test item-samples.

However applicant assessment indicates using the Cysteine 1:10 / Lysine 1:50 prediction model :

(i) the Mean of SPCC and SPCL depletion was in the > 6.38 < 22.62 - predicted low reactivity (Positive) class

(ii) the estimated result for lysine is suggested as possible: > 22.62 < 42.47 - predicted moderate reactivity (Positive) class

The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11-03-2020 to 02-05-2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-2 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-2 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422D – In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method and EURL ECVAM DB-ALM Protocol no. 155: KeratinoSens™, (Adopted March, 2018).
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.

PREPARATION OF TEST SOLUTIONS
- Other: A solubility test was performed. The test item was suspended in DMSO to a final concentration of 200 mM (clear colourless solution). The 100-fold dilution of the 200 mM DMSO stock formed a homogeneous solution (slight precipitation). The 100-fold dilution of the 12.5 to 100 mM DMSO stock also formed a homogeneous solution (microscopic precipitation). The 100-fold dilution of the 6.3 mM DMSO stock in DMEM glutamax formed also a homogeneous solution (no precipitation). 200 mM was selected as highest concentration for the first main assay (highest dose required in the current guideline).
- Preparation of the test chemical stock solution: In the main experiments the test item was dissolved in DMSO at 200 and 50 mM (colourless solution) in the first and second experiment, respectively. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (first experiment) and 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 μM (second experiment) (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. The test item precipitated at dose levels of 250 μM and above at the start and end of the incubation period, except in the second experiment, where precipitation at the start of the incubation period was only observed at 500 μM. Test item concentrations were used within 3 hours after preparation.
- Preparation of the test chemical serial dilutions: See above.
- Preparation of the positive controls: The positive control was Ethylene dimethacrylate glycol (EDMG), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted to the final concentration ranges of 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate. The PC formulation was used in other GLP studies concurrently to the present KERATINOSENS study.
- Preparation of the solvent, vehicle and negative controls : The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate. On each plate three blank wells were tested (no cells and no treatment).
- Stable dispersion obtained: Yes. Although, the test item precipitated at dose levels of 250 μM and above at the start and end of the incubation period, except in the second experiment, where precipitation at the start of the incubation period was only observed at 500 μM.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 μM (the highest test concentration required in the current guideline OECD 442C).
- Solubility in solvents: see ‘Preparation of test solutions’ ‘-Other’ for details on the solubility test.
- Solubility in incubation medium: Soluble up to maximum dose level. Although, the test item precipitated at dose levels of 250 μM and above at the start and end of the incubation period, except in the second experiment, where precipitation at the start of the incubation period was only observed at 500 μM.
- Cytotoxicity assessment performed: Yes. See below.
- Final concentration range selected on basis of: cytotoxicity (% viability) of the test item limited the concentration range. In experiment 1, the test item %viability was 85.1% at 31 μM and/or 6.6 at 63 μM. In experiment 2, the %viability was 97.4% at 31 μM and/or 8.1% at 63 μM. The concentration ranges were in accordance with the OECD TG 442D guideline including testing up to the highest dose required in the current guideline (2000 μM).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: Triplicate (3)
- Number of repetitions: Two (2) out of guideline specified three (3) with third only where applicable
- Test chemical concentrations: Final test concentrations of: Experiment 1: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%) and/or Experiment 2: 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 μM.
- Application procedure: See ‘cell culture and exposure’, below.
- Exposure time: 48 hours ± 1 h.
- Study evaluation and decision criteria used: See ‘Evaluation criteria’, below. In accordance with OECD 442C.
- Description on study acceptance criteria: See ‘Acceptability criteria’, below.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): See ‘cell culture and exposure’, below.
- Incubation conditions: See ‘cell culture and exposure’, below.
- Washing conditions: See ‘cell culture and exposure’, below.
- Precipitation noted: No precipitate was observed at any dose level tested.

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: TECAN Infinite® M200 Pro Plate Reade. The test system is fully validated. The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 1, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- Plate used: See ‘cell culture and exposure’, below.
- Lysate preparation: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from a recognised supplier were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady- Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
- Other: Not applicable.

DATA EVALUATION
- Cytotoxicity assessment: See ‘Cell Viability Assay MTT’, below.
- Prediction model used: See ‘Evaluation criteria’, below. In accordance with OECD 442C.
- Other: Not applicable.

- ACCEPTABILITY CRITERIA:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM). Actual PC: Experiment 1: 97 µM; Experiment 2: 56 µM.
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 97 µM and 56 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold (2.08-fold and 3.56-fold in experiment 1 and 2, respectively). Note: In the second experiment, the concentration of 31 μM already showed a luminesce induction of 1.56. However, since the concentration of 63 μM showed an induction of 1.48, the EC1.5 was calculated from the point were a clear dose related induction was observed.
(iii) average coefficient of variation (CoV) of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: Experiment 1: 6.5% ; Experiment 2: 9.0%.

CELL LINE USED
The KeratinoSens™ cell line is derived from the human keratinocyte culture HaCaT. It contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene. The cell line was developed by supplier (full details in the full study report). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE AND EXPOSURE
Cells are grown for 24 h in 96-well plates. The maintainece medium was Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL). Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock and were not cultured for more than 25 passages from the frozen stock (P+25). One day prior to testing cells were harvested and distributed into 96-well plates (10,000 cells/well) in basic medium (Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum). For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+9 in experiment 1 and P+10 in experiment 2. The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test item and control items were added. The exposure medium was Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum. Three wells per plate were left empty (no cells and no treatment) to assess background values.

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

- CELL VIABILITY ASSAY MTT
Test item IC50: IC50 value as the concentration in μM reducing the viability by 50%
Experiment 1: mean (n=3) 45 ; Experiment 2: mean (n=3) 48
Test item IC30: IC30 value as the concentration in μM reducing the viability by 30%
Experiment 1: mean (n=3) 37 ; Experiment 2: mean (n=3) 41
Conclusion:
The test item showed cytotoxicity (< 70% viability). The viability of the cells was lower than 70% within the concentration ranges both experiments and IC30 and IC50 values were calculated. The cytotoxic concentrations included at least one toxic concentration (< 70% viability) which was lower than the precipitating concentrations (250 μM and/or 500 μM, as applicable). Precipitation did not confound the conduct of the test assay.

- LUCIFERASE ASSAY
Imax indicating maximum fold-induction up to concentration 2000 μM
Experiment 1: mean (n=3) not determinable, the Imax was 1.39 and therefore no EC1.5 could be calculated ; and Experiment 2: mean (n=3) not determinable, the Imax was 1.39 and therefore no EC1.5 could be calculated

- DETERMINATIONS
EC1.5
Experiment 1: No dose related luminescence activity induction was observed after treatment with test item : EC1.5 = not applicable
Experiment 2: No dose related luminescence activity induction was observed after treatment with test item : EC1.5 = not applicable

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

RESULTS
2 out of 2 negative experiments (each in triplicate). The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) OR 0.24 – 500 µM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay. The test item showed toxicity (IC30 values of 37 μM and 41 μM and IC50 values of 45 μM and 48 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.29-fold and 1.32-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.
Applicant assessment indicates: the cytotoxic concentrations included at least one toxic concentration (< 70% viability) which was lower than the precipitating concentrations (250 μM and/or 500 μM, as applicable). Precipitation did not confound the conduct of the test assay.

Positive control results:

All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM). Actual PC: Experiment 1: 97 µM; Experiment 2: 56 µM.
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 97 µM and 56 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold (2.08-fold and 3.56-fold in experiment 1 and 2, respectively). Note: In the second experiment, the concentration of 31 μM already showed a luminesce induction of 1.56. However, since the concentration of 63 μM showed an induction of 1.48, the EC1.5 was calculated from the point were a clear dose related induction was observed.
(iii) average coefficient of variation (CoV) of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: Experiment 1: 6.5% ; Experiment 2: 9.0%.

Historical results for luciferase induction by the positive control in the test laboratory: average and standard deviations from 484 valid runs are presented in the full study report.

Key result

Run / experiment:

other: mean Experiment 1 (n = 3)

Parameter:

other: EC1.5

Remarks:

/ µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: mean Experiment 2 (n=3)

Parameter:

other: EC1.5

Remarks:

/ µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

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

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

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

- Acceptability criteria:
All acceptability criteria were met.
(i) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM). Actual PC: Experiment 1: 97 µM; Experiment 2: 56 µM.
(ii) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. Or if not achieved should give a satisfactory dose-response. The EC1.5 was 97 µM and 56 µM in experiment 1 and 2, respectively and the dose response in both experiments was greater than 2-fold (2.08-fold and 3.56-fold in experiment 1 and 2, respectively). Note: In the second experiment, the concentration of 31 μM already showed a luminesce induction of 1.56. However, since the concentration of 63 μM showed an induction of 1.48, the EC1.5 was calculated from the point were a clear dose related induction was observed.
(iii) average coefficient of variation (CoV) of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual: Experiment 1: 6.5% ; Experiment 2: 9.0%.

Historical results for luciferase induction by the positive control in the test laboratory: average and standard deviations from 484 valid runs are presented in the full study report.

Interpretation of results:

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

Conclusions:

Under the condition of this study, the test item is considered to be not sensitising to the skin. The test item gave 2 out of 2 negative experiments (each in triplicate) w The test item is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Executive summary:

The study was performed to the OECD TG 442D in vitro Skin Sensitisation guideline: ARE-Nrf2 Luciferase Test Method under GLP. The objective of this study was to evaluate the ability of the test item, to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in two independent experiments. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. The test item precipitated at dose levels of 250 μM and above at the start and end of the incubation period, except in the second experiment, where precipitation at the start of the incubation period was only observed at 500 μM. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between two standard deviations of the historical mean (97 µM and 56 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in experiment 2 (2.08-fold and 3.56-fold in experiment 1 and 2, respectively). It was noted for the second experiment, the concentration of 31 μM already showed a luminesce induction of 1.56. However, since the concentration of 63 μM showed an induction of 1.48, the EC1.5 was calculated from the point were a clear dose related induction was observed. The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 9.0% in experiment 1 and 2, respectively). In these experiments, the cells were incubated with the test item in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) or 0.24 – 500 µM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. The test item showed toxicity (IC30 values of 37 μM and 41 μM and IC50 values of 45 μM and 48 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.29-fold and 1.32-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM. All relevant test acceptability criteria were met.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13-10-2020 to 13-11-2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Well reported GLP study using a test method validated by the EURL ECVAM [test method: TM2011-09 (EU)] and included in the OECD Test Guideline Program (TGP no. 4.106). All relevant validity criteria were met. Test method considered to cover Key Event-3 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline Program (TGP no. 4.106)

Version / remarks:

Test protocol in accordance with the test method included in OECD Test Guideline Program (TGP no. 4.106).

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM test method: TM2011-09 (EU)

Version / remarks:

Test protocol in accordance with the test method included in EURL ECVAM validation

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381

Version / remarks:

Test method as described in the aforementioned publication and related literature citations

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169

Version / remarks:

Test method as described in the aforementioned publication and related literature citations

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: A. Forreryd et. al. (2016), From genome-wide arrays to tailor-made biomarker readout – Progress towards routine analysis of skin sensitizing chemicals with GARD, Toxicology in Vitro 37, 178–188

Version / remarks:

Test method as described in the aforementioned publication and related literature citations

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Principles of method if other than guideline:

Well reported GLP study using a test method validated by the EURL ECVAM [test method: TM2011-09 (EU)] and included in the OECD Test Guideline Program (TGP no. 4.106). All relevant validity criteria were met. Test method considered to cover Key Event-3 under OECD 168 (2014) and OECD 255 (2017) and OECD 256 (2017).

Prior to the test assay: the test item was assessed for solubility and cytotoxic effect in order to establish the GARD input concentration (concentration inducing 90% relative viability):
(1) Solubility testing was performed to identify the most appropriate vehicle for the test item. Under the test method: the test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a test system compatible vehicle to a minimum in-well concentration of 1 μM. For the test item, the maximum target in-well concentration of any test item was defined to be 500 μM. If the test item was not soluble to the maximum in-well concentration of 500 μM, the vehicle that generated the highest test item in-well concentration is utilised, with a minimum recommended in-well concentration of 1 μM. The maximum assessed concentration in vehicle was 500 mM in DMSO. Based on solubility testing, the vehicle chosen for downstream procedures was DMSO (0.1%) and the highest soluble in well concentration was determined to 500 μM.
(2) Cytotoxicity testing was performed between 500 μM and 1 μM in-well concentration. The results of the cytotoxicity assessment indicated cytotoxicity (see table 1). The Rv90 was shown to be between 50 μM and 100 μM in-well concentration. Relative Viability (%) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item.

Additional notes applicable to ‘difficult test items’:
The applicant is aware of an acceptable standard protocol deviation that may be used to enhance the sensitivity of the test assay for difficult test items which show solubility limitations and show little to no cytotoxicity. Therein, the vehicle diluted test Items are directly diluted (1-step dilution) in the cell medium in order to assess the highest (potential) GARD assay "in-well concentration". The cell medium diluted test item is further transferred to the SenzaCells for stimulation according to the Standard GARD Assay Protocol.
Basis:
(i) Where the test Item shows solubility limitations in cell medium when using the Standard GARD Assay Protocol, a 1-step dilution in vehicle may be used to increase the in-well concentration to the upper limit of the titration range.
(ii) Where no cellular cytotoxicity is observed, the GARD input concentration for the test item is determined to 500 µM. Input concentration would only be limited by cytotoxicity considerations.

References:
(1) H. Johansson et al., The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27 (2013) 1163–1169
(2) Test system manufacturer: [Senzagen AB, Sweden] published research (2019) indicates: DMSO (0.1%), water and/or Acetone, DMF, DMF/Glycerol, Ethanol, Glycerol, Isopropanol have been determined all compatible solvents with the test system, the latter of which are used increase its applicability domain (by increasing test item solubility) when testing difficult to dissolve test items: Applicant assessment indicates these solvent options are likely to be incorporated in future OECD TG publication, and/or have been demonstrated by the manufacturer to be compatible with the test system.

This standard protocol deviation would be used to enhance the sensitivity of the test assay by maximising concentration/exposure of the test item to the SenzaCell : human myeloid leukemia-derived cell line by utilising the highest soluble concentration and/or 500 µM concentration, where possible in the absence of cytotoxicity and a determinable Rv90 (relative viability 90%).These dilution and solvent options are likely to be incorporated in future OECD TG publication, and/or have been demonstrated by the manufacturer to be compatible with the test system.

Since the test item was fully soluble to 500 μM in DMSO (0.1%) this protocol deviation was not required in the present study. A standard vehicle, DMSO (0.1%) was also utilised.

GLP compliance:

yes

Remarks:

The laboratory where the study was conducted (details given in the full study report) achieved GLP certification: 26 May 2020 (inspected 06 to 15 April 2020). Information in the public domain.

Type of study:

activation of dendritic cells

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
- The study was conducted in accordance with the GARDskin assay protocol ; non-exhaustive references include: OECD Test Guideline Program (TGP no. 4.106), and which has been EURL ECVAM validated according to test method: TM2011-09 (EU), and with reference to the non-exhaustive related publications: (1) H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381 ; and/or : (2) H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169. The test assay is pending full OECD acceptance. The laboratory where the study was conducted (details given in the full study report) achieved GLP certification: 26 May 2020 (inspected 06 to 15 April 2020). Information in the public domain.
- The study protocol was previously validated (in-house laboratory and/or external validations) with reference chemicals described in the published literature. The results of the testing on the reference chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). Additional public information on the in-house validation can be found in: H. Johansson et al. (2014), Genomic allergen rapid detection in-house validation--a proof of concept, Toxicol. Sci. ; 139 (2) : 362-70 and/or H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169 ; and/or for external validation within: H. Johansson et al., (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381. Additional information is in the public domain on the EURL ECVAM and OECD websites.

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The maximum assessed concentration in vehicle was 500 mM in DMSO. The vehicle chosen for downstream procedures was DMSO and the highest soluble in well concentration was determined to be 500 μM.
- Preparation of the test chemical serial dilutions: Not relevant. Only a single ‘GARD input concentration’ is utilised which is the maximum soluble concentration on the basis of properties and/or cytotoxicity. A 2-step dilution is performed at the ‘GARD input concentration’ : “for cell stimulations, test items are dissolved in its appropriate solvent as 1000x stocks of target in-well concentration, called “stock A”. A 10x stock, called “stock B”, is prepared by taking 10 μL of stock A to 990 μL of cell media. 200 μL of stock B is then added to the wells containing 1.8 mL seeded cells. For the samples dissolved in DMSO, the in-well concentration of DMSO will thus be 0.1% (sic)”. Reference: H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169. See “dose range finding assay” for further information. Further enhancements can be made to increase solubility for ‘difficult test items’. Through choice of 1-step dilution direct into test medium and/or alternative test system compatible solvents. See “Other:” below.
- Preparation of the positive controls: The positive control : p-Phenylenediamine (PPD) was prepared analogous to the test item (see above) and at 75 μM in-well concentration in DMSO (0.1%).
- Preparation of the solvent, vehicle and negative controls: The negative control : DMSO (0.1%) was directly applied to the test system.
- Stable dispersion obtained: Yes.
- Log Kow of the test chemical: Not relevant. The test system does not have log Kow limitations. [Log Kow of the test item: 5.6, (Anon., OECD TG 117, 2020)]. Further enhancements can be made to increase solubility for ‘difficult test items’. Through choice of 1-step dilution direct into test medium and/or alternative test system compatible solvents. See “Other:” below.
- Other: GARDskin assay does not have the limitations of other OECD TG 442E methods (such as h-CLAT). See other comments below.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 500 μM (in-well concentration)
- Solubility in solvents: Solubility testing was performed to identify the most appropriate vehicle for the test item. The maximum assessed concentration in vehicle was 500 mM in DMSO. Under the test method: the test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a test system compatible vehicle to a minimum in-well concentration of 1 μM. For the test item, the maximum target in-well concentration of any test item was defined to be 500 μM. If the test item was not soluble to the maximum in-well concentration of 500 μM, the vehicle that generated the highest test item in-well concentration is utilised, with a minimum recommended in-well concentration of 1 μM. Based on solubility testing, the vehicle chosen for downstream procedures was DMSO (0.1%) and the highest soluble in well concentration was determined to 500 μM.
- Solubility in incubation medium: No issues reported.
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: Cytotoxicity testing was performed between 500 μM and 1 μM in-well concentration. The results of the cytotoxicity assessment indicated cytotoxicity (see table 1). The Rv90 was shown to be between 50 μM and 100 μM in-well concentration. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item. Relative Viability (%) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%.
- Final concentration range selected on basis of: Cytotoxicity. “For cytotoxic test items, the concentration yielding 90% relative viability (Rv90) should be used for the GARD assay. For non-cytotoxic test items, a concentration of 500 µM should be used if possible. For non-toxic compounds that are insoluble at 500 µM in cell media, the highest soluble concentration should be utilised. Whichever of these three criteria is met, only one concentration will be used for the genomic assay. The concentration to be used for any given chemical is termed the ‘GARD input concentration’ (sic)”. Reference: H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169.
- Other: Additional notes applicable to ‘difficult test items’:
The applicant is aware of an acceptable standard protocol deviation that may be used to enhance the sensitivity of the test assay for difficult test items which show solubility limitations and show little to no cytotoxicity. Therein, the vehicle diluted test Items are directly diluted (1-step dilution) in the cell medium in order to assess the highest (potential) GARD assay "in-well concentration". The cell medium diluted test item is further transferred to the SenzaCells for stimulation according to the Standard GARD Assay Protocol.
Basis:
(i) Where the test Item shows solubility limitations in cell medium when using the Standard GARD Assay Protocol, a 1-step dilution in vehicle may be used to increase the in-well concentration to the upper limit of the titration range.
(ii) Where no cellular cytotoxicity is observed, the GARD input concentration for the test item is determined to 500 µM. Input concentration would only be limited by cytotoxicity considerations.
References:
(1) H. Johansson et al., The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27 (2013) 1163–1169
(2) Test system manufacturer: [Senzagen AB, Sweden] published research (2019) indicates: DMSO (0.1%), water and/or Acetone, DMF, DMF/Glycerol, Ethanol, Glycerol, Isopropanol have been determined all compatible solvents with the test system, the latter of which are used increase its applicability domain (by increasing test item solubility) when testing difficult to dissolve test items: Applicant assessment indicates these solvent options are likely to be incorporated in future OECD TG publication, and/or have been demonstrated by the manufacturer to be compatible with the test system.

This standard protocol deviation would be used to enhance the sensitivity of the test assay by maximising concentration/exposure of the test item to the SenzaCell : human myeloid leukaemia-derived cell line by utilising the highest soluble concentration and/or 500 µM concentration, where possible in the absence of cytotoxicity and a determinable Rv90 (relative viability 90%). These dilution and solvent options are likely to be incorporated in future OECD TG publication, and/or have been demonstrated by the manufacturer to be compatible with the test system.

Since the test item was fully soluble to 500 μM in DMSO (0.1%) this protocol deviation was not required in the present study. A standard vehicle, DMSO (0.1%) was also utilised.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: Triplcate (3)
- Number of repetitions: Single (1)
- Test chemical concentrations: 500 μM (in-well concentration) = ‘GARD input concentration’
- Application procedure: In the main experiment(s) the cells are stimulated (as previously described) using the GARD input concentration. In addition to the assayed test Item a set of concurrent positive and negative controls are performed as reference and quality controls. Test item and controls were assayed in biological replicates (n=3). After incubation for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity, cell culture was lysed in TRizol reagent and stored at -20 ± 5 °C until RNA was extracted/isolated. In parallel, stimulated cells were PI stained and analysed by flow cytometry to verify the expected relative viability. RNA isolation from lysed cells was performed using commercially available kits (referenced in the full study report). Total RNA was quantified, and quality controlled using BioAnalyzer equipment.
- Exposure time: incubation for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity (then cell culture lysis)
- Study evaluation and decision criteria used: Endpoint measurement:
(i) Gene expression analysis using Nanostring nCounter System : A total of 100 ng of RNA was used as sample input in a hybridization assay with the GPS (GARD Prediction Signature) specific Reporter CodeSet. The hybridized sample was prepared on chip using nCounter Preparation Station and individual transcripts of the GPS were quantified using Digital Analyzer. Data was imported into relevant software environment for statistical computation analysis (documented in the full study report). Raw data was normalised using a gene specific Counts Per Total Counts (CPTC) algorithm.
(ii) Prediction model and evaluation: For assessment of skin sensitisation, the Support Vector Machine (SVM) was modelled on a training data set corresponding to samples used for assay development. For a comprehensive overview of the training data set and methods, this is referenced in the full study report. Batch variations between the training data set and the test data set were eliminated using the Batch Adjustment by Reference Alignment (BARA) method, using unstimulated cells as a reference control. Each sample in the test set was assigned a Decision Value (DV) based on its transcriptional levels of the endpoint specific biomarker signature.

EVALUATION CRITERIA:
WITHIN GARDSKIN:
Each sample in the test set were assigned a Support Vector Machine (SVM) Decision Value (DV), based on its transcriptional levels of the GPS biomarker signature.
Test item considered ‘negative’ where: mean DV < 0 (n ≥ 2) which results in no classification as a skin sensitiser (or ‘non-sensitising’).
Test item considered ‘positive’ where: mean DV ≥ 0 (n ≥ 2) which results in classification as a skin sensitiser.
WITHIN GARDPOTENCY:
For potency classification, a test item assigned a median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A). Consequently, a test item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B).
Further information on GARDpotency sub-model is provided separately.

- Description on study acceptance criteria: All applicable acceptability criteria were met.
(1) Positive Control: p-phenvlendiamine (PPD) [CAS 106-50-3] gave a positive prediction for sensitisation (passed GARDskin Assay Quality Control) and/or accurately classified.
(2) Negative Control: dimethyl sulfoxide (DMSO) [CAS 67-68-5] gave a negative prediction for sensitisation (passed GARDskin Assay Quality Control) and/or accurately classified.
(3) The test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a GARDskin and GARDpotency compatible vehicle to a minimum in-well concentration of 1 μM. (Unless standard protocol deviations utilised ; see above).
(4) The Main Stimulation cells should pass the Phenotypic Quality Control on the day of cell stimulation and the acceptance criteria for specified biomarker profile(s) for: CD86 (10-40%) ; CD54 (+ presence) ; HLA-DR (+ presence) ; CD80 (≤ 10%) ; CD34 (+ presence) ; CD14 (+ presence) ; CD1a (+ presence).
(5) The cell viability of the Main Stimulations should have fulfilled the following acceptance criteria: The positive control and a test item expected to induce cytotoxicity, should have a relative viability of 84.5 % - 95.4 %. The negative control and a test item not expected to induce cytotoxicity, should have a relative viability of ≥ 95.5 %. The unstimulated control should have an absolute viability of ≥ 84.5 %.
(6) For the RNA Quality Control : the total RNA concentration ≥ 20 ng/μL and/or a RIN value ≥ 8 and this should be observed in at least two test item replicates. For unstimulated (negative) controls and/or positive controls: individual replicates should pass.
(7) For the endpoint measurement quality control: the RCC files generated from the nCounter MAX analysis files should fulfil the following acceptance criteria: Imaging Quality: > 0.75 ; Linearity: > 0.95 ; Limit of Detection (LOD): (POS_E / LOD) > 1 and/or Binding Density (BDI: 0.05 < 80 < 2.25) and this should be observed in at least two test item replicates. For unstimulated (negative) controls and/or positive controls: individual replicates should pass.
- Other: No precipitation was reported observed at any point during the study including the start and end of the incubation period in solutions and wells.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): For cytotoxicity assessment, cells at passage number 4-16 were used. For Main Stimulation, cells at passage number 6-12 were utilised.
- Incubation conditions: For cytotoxicity assessment: 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity. For chemical stimulation of cells, exposed cells are incubated for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity.
- Washing conditions: After incubation for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2, the cells were lysed in TRlzol reagent and stored at -20 ± 5 °C awaiting RNA isolation. In parallel, cells from the same stimulation well were PI stained and analysed by flow cytometry as described in Cytotoxicity Assessment to verify the expected relative viability.
- Other: Details on and maintainece of the test cell line: The human myeloid leukaemia-derived cell line SenzaCell (available through ATCC), acting as an in vitro model of human Dendritic Cell (DC), is maintained in a-MEM supplemented with 20% (volume/volume) fetal calf serum and 40 ng/mL recombinant human Granulocyte Macrophage Colony Stimulating Factor (rhGM-CSF). During cell propagation, subculturing with fresh medium was performed every 2-3 days to a concentration of 0.2x10^6 cells/mL. Working stocks of cultures were grown for a maximum of 16 passages or two months after thawing.
Cells were seeded for stimulation directly following a cell split, i.e. the test item stimulations were scheduled to coincide with routine cell culture maintenance. The cell stimulations were initiated when a stable cell culture was established i.e. when at least a duplication of the cells between cell passages was seen, and, depending on the purpose of the cell stimulation, at specific cell passage ranges (see above).

DATA EVALUATION
- Cytotoxicity assessment: A test item that induces cytotoxicity was used at the concentration that induces 90 ± 5 % relative viability. This concentration ensures bioavailability of the test item, while not impairing immunological responses. (For information only: a test item that was not cytotoxic (e.g. relative viability ca. 95%] would be tested at a concentration of 500 μM, or at the highest soluble concentration).
- Prediction model used: Within GARDskin:
Each sample in the test set were assigned a Support Vector Machine (SVM) Decision Value (DV), based on its transcriptional levels of the GPS biomarker signature. Test item considered ‘negative’ where: mean DV < 0 (n ≥ 2) which results in no classification as a skin sensitiser (or ‘non-sensitising’). Test item considered ‘positive’ where: mean DV ≥ 0 (n ≥ 2) which results in classification as a skin sensitiser.
Within GARDpotency:
For potency classification, a test item assigned a median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A). Consequently, a test item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B).
Further information on GARDpotency sub-model is provided separately.
- Other: Further information is provided in ‘any other information on results including tables’.

RESULTS:
The mean SVM DV was +2.02, the GARDskin Prediction was “sensitising”. All relevant concurrent positive and negative controls passed GARDskin Assay Quality Control criteria. All relevant validity criteria were met.
Further information on GARDpotency sub-model is provided separately.

Positive control results:

Positive Control: p-phenvlendiamine (PPD) [CAS 106-50-3] gave a positive prediction for sensitisation (PC SVM Decision Value > 0) and passed GARDSkin Assay Quality Control

Key result

Run / experiment:

other: mean (n=3)

Parameter:

other: GARDSkin SVM Decision Value

Remarks:

TEST ITEM

Value:

2.02

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

mean SVM DV = -0.989 (non-sensitiser)

Positive controls validity:

valid

Remarks:

mean SVM DV = +8.21 (sensitiser)

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported. The results of the cytotoxicity assessment indicated cytotoxicity (see table 1). The Rv90 was shown to be between 50 μM and 100 μM in-well concentration. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item. Relative Viability (%) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study was conducted in accordance with the GARDskin assay protocol ; non-exhaustive references include: OECD Test Guideline Program (TGP no. 4.106), and which has been EURL ECVAM validated according to test method: TM2011-09 (EU), and with reference to the non-exhaustive related publications: (1) H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381 ; and/or : (2) H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169. The test assay is pending full OECD acceptance. The laboratory where the study was conducted (details given in the full study report) achieved GLP certification: 26 May 2020 (inspected 06 to 15 April 2020). Information in the public domain. The study protocol was previously validated (in-house laboratory and/or external validations) with reference chemicals described in the published literature. The results of the testing on the reference chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). Additional public information on the in-house validation can be found in: H. Johansson et al. (2014), Genomic allergen rapid detection in-house validation--a proof of concept, Toxicol. Sci. ; 139 (2) : 362-70 and/or H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169 ; and/or for external validation within: H. Johansson et al., (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381. Additional information is in the public domain on the EURL ECVAM and OECD websites. Further information on GARDpotency sub-model is provided separately.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: No issues were reported in the full study report.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria: All applicable acceptability criteria were met. Further information is provided in ‘any other information on results including tables’.
(1) Positive Control: p-phenvlendiamine (PPD) [CAS 106-50-3] gave a positive prediction for sensitisation (passed GARDskin Assay Quality Control) and/or accurately classified.
(2) Negative Control: dimethyl sulfoxide (DMSO) [CAS 67-68-5] gave a negative prediction for sensitisation (passed GARDskin Assay Quality Control) and/or accurately classified.
(3) The test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a GARDskin and GARDpotency compatible vehicle to a minimum in-well concentration of 1 μM. (Unless standard protocol deviations utilised ; see above).
(4) The Main Stimulation cells should pass the Phenotypic Quality Control on the day of cell stimulation and the acceptance criteria for specified biomarker profile(s) for: CD86 (10-40%) ; CD54 (+ presence) ; HLA-DR (+ presence) ; CD80 (≤ 10%) ; CD34 (+ presence) ; CD14 (+ presence) ; CD1a (+ presence).
(5) The cell viability of the Main Stimulations should have fulfilled the following acceptance criteria: The positive control and a test item expected to induce cytotoxicity, should have a relative viability of 84.5 % - 95.4 %. The negative control and a test item not expected to induce cytotoxicity, should have a relative viability of ≥ 95.5 %. The unstimulated control should have an absolute viability of ≥ 84.5 %.
(6) For the RNA Quality Control : the total RNA concentration ≥ 20 ng/μL and/or a RIN value ≥ 8 and this should be observed in at least two test item replicates. For unstimulated (negative) controls and/or positive controls: individual replicates should pass.
(7) For the endpoint measurement quality control: the RCC files generated from the nCounter MAX analysis files should fulfil the following acceptance criteria: Imaging Quality: > 0.75 ; Linearity: > 0.95 ; Limit of Detection (LOD): (POS_E / LOD) > 1 and/or Binding Density (BDI: 0.05 < 80 < 2.25) and this should be observed in at least two test item replicates. For unstimulated (negative) controls and/or positive controls: individual replicates should pass.
Further information on GARDpotency sub-model is provided separately.

Table 1. Test item cytotoxicity assessment:

	Relative viability (%)
In-well concentration (µM)	100	90	80	70	60	50
Test item #1 replicate	90.20	85.35	91.04	93.52	95.80	95.54
Test item #2 replicate	87.92	91.67	92.97	96.36	95.52	97.55
Test item #3 replicate	89.79	91.26	92.11	94.23	95.82	96.75

 

Table 2. Relative viability of Test and Reference Items and the result of the RNA Quality Controls .

Test/Reference Item	Replicate	Stimulation conc (i)	Vehicle	Relative viability (ii) (%)	Acceptance criteria (Pass/Fail)	RNA conc (ng/µL)	RIN	RNA QC (Pass/Fail)
Test Item	1	100 µM	DMSO	90.20	Pass	22	9.8	Pass
	2	100 µM	DMSO	87.92	Pass	22	10.0	Pass
	3	100 µM	DMSO	89.79	Pass	50	10.0	Pass
Positive control (PPD)	1	75 µM	DMSO	87.66	Pass	74	9.8	Pass
	2	75 µM	DMSO	88.13	Pass	48	9.7	Pass
	3	75 µM	DMSO	91.21	Pass	69	10.0	Pass
Negative control (DMSO)	1	0.1 %	Not applicable	100.05	Pass	39	9.9	Pass
	2	0.1 %	Not applicable	99.43	Pass	23	10.0	Pass
	3	0.1 %	Not applicable	100.10	Pass	22	9.8	Pass
Unstimulated control (absolute viability %)	1	Not applicable	Not applicable	96.50 (iii)	Pass	28	10.0	Pass
	2	Not applicable	Not applicable	96.05 (iii)	Pass	36	10.0	Pass
	3	Not applicable	Not applicable	97.00 (iii)	Pass	40	9.9	Pass

(i) Concentration based on max. solubility and cytotoxicity.

(ii) Observed viability, calculated based on the equation in section Standard GARDskin Dose-Response Assay Procedure.

(iii) Absolute Viability

 

Table 3. Result of the Endpoint measurement Quality Control for the GARDskin assessment.

Test/Reference Item	Replicate	Imaging Quality	Binding Density	Limit of detection	Linearity	Endpoint measurement QC (Pass/Fail)
Test item	1	0.95	0.49	11.35	0.999	Pass
	2	0.96	0.46	8.81	0.998	Pass
	3	0.96	0.50	13.48	0.999	Pass
Positive control (PPD)	1	0.96	0.45	9.078	0.997	Pass
	2	0.95	0.48	12.03	0.998	Pass
	3	0.96	0.48	12.92	0.998	Pass
Negative control (DMSO)	1	0.94	0.45	11.54	0.999	Pass
	2	0.95	0.48	14.08	0.999	Pass
	3	0.96	0.38	10.99	0.996	Pass
Unstimulated control	1	0.93	0.51	11.63	0.998	Pass
	2	0.94	0.45	11.60	0.998	Pass
	3	0.96	0.43	10.68	0.996	Pass

 

Table 4. Result of the Endpoint measurement Quality Control for the GARDpotency assessment.

Test/Reference Item	Replicate	Imaging Quality	Binding Density	Limit of detection	Linearity	Endpoint measurement QC (Pass/Fail)
Test item	1	0.97	0.29	4.83	0.998	Pass
	2	0.97	0.28	5.59	0.999	Pass
	3	0.98	0.30	4.66	0.997	Pass
Positive control (PPD)	1	0.97	0.23	4.95	0.997	Pass
	2	0.98	0.26	4.67	0.998	Pass
	3	0.97	0.27	4.85	0.998	Pass
Unstimulated control	1	0.98	0.30	4.66	0.997	Pass
	2	0.97	0.28	5.20	0.996	Pass
	3	0.94	0.24	4.61	0.998	Pass

 

Table 5.Table12: Decision Values and GARDskin classifications.

Test/Reference Item	Decision value (DV)			Mean DV	GARDskin classification	Acceptance criteria (i) (Pass/Fail)
	Replicate #1	Replicate #2	Replicate #3
Test item	+1.32	+2.75	+2.01	+2.02	Sensitizer	Not applicable for Test Item
Positive control (PPD)	+7.88	+7.82	+8.94	+8.21	Sensitizer	Pass
Negative control (DMSO)	-1.18	-1.37	-0.419	-0.989	Non-sensitizer	Pass

(i) Reference Item acceptance criteria as described in section Standard GARD Assay Acceptance Criteria.

 

Table 6: Decision Values and GARDpotency classifications.

Test/Reference Item	Decision value (DV)			Median DV	GARDpotency classification	Acceptance criteria (i) (Pass/Fail)
	Replicate #1	Replicate #2	Replicate #3
Test item	-0.265	-0.954	-0.915	-0.915	UN GHS SS 1B	Not applicable for Test Item
Positive control (PPD)	+3.70	+4.35	+4.53	+4.35	UN GHS SS 1A	Pass

(i) Reference Item acceptance criteria as described in section Standard GARD Assay Acceptance Criteria.

Interpretation of results:

other: The test item gave a positive result based on the mean SVM DV > 0 (test conducted in triplicate). The result will be considered within a weight of evidence assessment for C&L purposes.

Remarks:

Further information on GARDpotency sub-model is provided separately.

Conclusions:

Under the condition of this study, the test item is considered to be sensitising to the skin. The test item gave a mean Support Vector Machine (SVM) Decision Value (DV) of +2.02. The GARDskin prediction was sensitising since the mean SVM DV > 0 (n ≥ 2). All relevant concurrent positive and negative controls passed GARDskin Assay Quality Control criteria. All applicable acceptability criteria were met.

Executive summary:

The study was performed to the GARDskin (Genomic Allergen Rapid Detection) test method which has been EURL ECVAM validated [test method: TM2011-09 (EU)] and is included in the OECD Test Guideline Program (TGP no. 4.106), pending full OECD TG adoption. The test assay is detailed in the following non-exhaustive related publications: (1) H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381 ; and/or : (2) H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169 ; and/or (3) A. Forreryd et. al. (2016), From genome-wide arrays to tailor-made biomarker readout – Progress towards routine analysis of skin sensitizing chemicals with GARD, Toxicology in Vitro 37, 178–188 and/or (4) R. Gradin et al. (2020) The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432. The GARD platform is a versatile testing strategy that relies on gene expression analysis of cell cultures exposed to test items in vitro. The high-dimensional readout allows for gene expression analysis of customized biomarker signatures, each specific for various biological end points. In addition, the final prediction gives rise to the classification of test items based on a statistical prediction model that is in turn dependent on an end point specific training data set as to if a substance is skin sensitising or non-skin sensitising. The human myeloid leukaemia-derived cell line SenzaCell (available through ATCC), acting as an in vitro model of human Dendritic Cell (DC), is maintained in a-MEM supplemented with 20% (volume/volume) fetal calf serum and 40 ng/mL recombinant human Granulocyte Macrophage Colony Stimulating Factor (rhGM-CSF). During cell propagation, subculturing with fresh medium was performed every 2-3 days to a concentration of 0.2x10^6 cells/mL. Working stocks of cultures were grown for a maximum of 16 passages or two months after thawing. Cells were seeded for stimulation directly following a cell split, i.e. the test item stimulations were scheduled to coincide with routine cell culture maintenance. The cell stimulations were initiated when a stable cell culture was established i.e. when at least a duplication of the cells between cell passages was seen, and, depending on the purpose of the cell stimulation, at specific cell passage ranges. For cytotoxicity assessment, cells at passage number 4-16 were used. For Main Stimulation, cells at passage number 6-12 were utilised. To verify that cells were maintained in an inactivated state, and to detect potential phenotypic drift, the cells were stained with a panel of biomarkers, CD54, CD86, HLA-DR, CD34, CDS0, CD14 and CD1a. For chemical stimulation of cells, exposed cells are incubated for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity. Prior to the assay: the test item was assessed for solubility and cytotoxic effect in order to establish the GARD input concentration (concentration inducing 90% relative viability). Cytotoxicity testing was performed between 500 μM and 1 μM in-well concentration. Solubility testing was performed to identify the most appropriate vehicle for the test item. The maximum assessed concentration in vehicle was 500 mM in DMSO. Under the test method: the test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a test system compatible vehicle to a minimum in-well concentration of 1 μM. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item. Relative Viability (%) (mean ; n=3 replicates) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%. In the main experiment(s) the cells are stimulated (as previously described) using the GARD input concentration. In addition to the assayed test Item a set of concurrent positive and negative controls are performed as reference and quality controls. Test item and controls were assayed in biological replicates (n=3). After incubation for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity, cell culture was lysed in TRizol reagent and stored at -20 ± 5 °C until RNA was extracted/isolated. In parallel, stimulated cells were PI stained and analysed by flow cytometry to verify the expected relative viability. RNA isolation from lysed cells was performed using commercially available kits (referenced in the full study report). Total RNA was quantified, and quality controlled using BioAnalyzer equipment. The test item indicated no cellular cytotoxicity, therefore the GARD input concentration was determined to 100 µM for the test item. The concurrent positive control was p-phenvlendiamine (PPD) [CAS 106-50-3], for which an in-well concentration of 75 µM in DMSO was utilised. The concurrent negative control was dimethyl sulfoxide (DMSO) [CAS 67-68-5], for which an in-well concentration of 0.10% was utilised. Under the condition of this study, the test item is considered to be sensitising to the skin. The test item gave a mean Support Vector Machine (SVM) Decision Value (DV) of +2.02. The GARDskin prediction was sensitising since the mean SVM DV > 0 (n ≥ 2). Under the separate GARDpotency sub-model, the prediction was UN GHS Skin Sensitiser 1B as the median SVM DV was -0.915 since a test item assigned with median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A) and a test item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B). Further information on GARDpotency sub-model is provided separately. The positive and negative controls gave acceptable results. The phenotypic quality control, the cytotoxicity/relative viability quality controls, the RNA quality control and the endpoint measurement quality controls all met the acceptance criteria. All applicable acceptability criteria were considered to be met.

Reference 4

Endpoint:

skin sensitisation: in vitro

Remarks:

GARDpotency assay : used to discriminate between UN GHS sub-category 1A skin sensitizers and a combination of sub-category 1 B and non-sensitizers according to UN GHS classification criteria.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13-10-2020 to 13-11-2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Well reported GLP study using GARDpotency test method/model validated by the EURL ECVAM [test method: TM2011-09 (EU)] and included in the OECD Test Guideline Program (TGP no. 4.106). GARDpotency supplemental information is provided in the attached summary.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline Program (TGP no. 4.106)

Version / remarks:

Test protocol in accordance with the test method included in OECD Test Guideline Program (TGP no. 4.106).

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM test method: TM2011-09 (EU)

Version / remarks:

Test protocol in accordance with the test method included in EURL ECVAM validation

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: R. Gradin et al. (2020) The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432

Version / remarks:

Test method as described in the aforementioned publication and related literature citations

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381

Version / remarks:

Test method as described in the aforementioned publication and related literature citations

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169

Version / remarks:

Test method as described in the aforementioned publication and related literature citations

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Qualifier:

according to guideline

Guideline:

other: A. Forreryd et. al. (2016), From genome-wide arrays to tailor-made biomarker readout – Progress towards routine analysis of skin sensitizing chemicals with GARD, Toxicology in Vitro 37, 178–188

Version / remarks:

Test method as described in the aforementioned publication and related literature citations

Deviations:

no

Remarks:

see "Principles of method if other than guideline" for further information

Principles of method if other than guideline:

Well reported GLP study using GARDskin and GARDpotency test methods/models validated by the EURL ECVAM [test method: TM2011-09 (EU)] and included in the OECD Test Guideline Program (TGP no. 4.106). GARDpotency supplemental information is provided within the following literature reference:

R. Gradin et al. (2020) The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432

For the main GARDskin test method and model refer to citations in “test guideline”.

Additionally, further information on GARDskin main test assay and model is provided separately in the attached summary (see “cross-reference” - “reference to same study”).

Prior to the test assays: the test item was assessed for solubility and cytotoxic effect in order to establish the GARD input concentration (concentration inducing 90% relative viability):
(1) Solubility testing was performed to identify the most appropriate vehicle for the test item. Under the test method: the test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a test system compatible vehicle to a minimum in-well concentration of 1 μM. For the test item, the maximum target in-well concentration of any test item was defined to be 500 μM. If the test item was not soluble to the maximum in-well concentration of 500 μM, the vehicle that generated the highest test item in-well concentration is utilised, with a minimum recommended in-well concentration of 1 μM. The maximum assessed concentration in vehicle was 500 mM in DMSO. Based on solubility testing, the vehicle chosen for downstream procedures was DMSO (0.1%) and the highest soluble in well concentration was determined to 500 μM.
(2) Cytotoxicity testing was performed between 500 μM and 1 μM in-well concentration. The results of the cytotoxicity assessment indicated cytotoxicity (see table 1). The Rv90 was shown to be between 50 μM and 100 μM in-well concentration. Relative Viability (%) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item.

GLP compliance:

yes

Remarks:

The laboratory where the study was conducted (details given in the full study report) achieved GLP certification: 26 May 2020 (inspected 06 to 15 April 2020). Information in the public domain.

Type of study:

activation of dendritic cells

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
- The GARDskin main test assay was conducted in accordance with the GARDskin assay protocol ; non-exhaustive references include: OECD Test Guideline Program (TGP no. 4.106), and which has been EURL ECVAM validated according to test method: TM2011-09 (EU), and with reference to the non-exhaustive related publications: (1) H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381 ; and/or : (2) H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169. The test assay is pending full OECD acceptance. The laboratory where the study was conducted (details given in the full study report) achieved GLP certification: 26 May 2020 (inspected 06 to 15 April 2020). Information in the public domain.
- The study protocol was previously validated (in-house laboratory and/or external validations) with reference chemicals described in the published literature. The results of the testing on the reference chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). Additional public information on the in-house validation can be found in: H. Johansson et al. (2014), Genomic allergen rapid detection in-house validation--a proof of concept, Toxicol. Sci. ; 139 (2) : 362-70 and/or H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169 ; and/or for external validation within: H. Johansson et al., (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381. Additional information is in the public domain on the EURL ECVAM and OECD websites.

- The sub-test assay/sub-model: GARDpotency: is a related model – which has also been submitted for EURL ECVAM validated according to test method: TM2011-09 (EU) and/or is included in OECD Test Guideline Program (TGP no. 4.106). The main literature citation is : (4) R. Gradin et al. (2020) The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The maximum assessed concentration in vehicle was 500 mM in DMSO. The vehicle chosen for downstream procedures was DMSO and the highest soluble in well concentration was determined to be 500 μM.
- Preparation of the test chemical serial dilutions: Not relevant. Only a single ‘GARD input concentration’ is utilised which is the maximum soluble concentration on the basis of properties and/or cytotoxicity. A 2-step dilution is performed at the ‘GARD input concentration’ : “for cell stimulations, test items are dissolved in its appropriate solvent as 1000x stocks of target in-well concentration, called “stock A”. A 10x stock, called “stock B” ,is prepared by taking 10 μL of stock A to 990 μL of cell media. 200 μL of stock B is then added to the wells containing 1.8 mL seeded cells. For the samples dissolved in DMSO, the in-well concentration of DMSO will thus be 0.1% (sic)”. Reference: H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169. See “dose range finding assay” for further information. Further enhancements can be made to increase solubility for ‘difficult test items’. Through choice of 1-step dilution direct into test medium and/or alternative test system compatible solvents. See “Other:” below.
- Preparation of the positive controls: The positive control : p-Phenylenediamine (PPD) was prepared analogous to the test item (see above) and at 75 μM in-well concentration in DMSO (0.1%).
- Preparation of the solvent, vehicle and negative controls: The negative control : DMSO (0.1%) was directly applied to the test system.
- Stable dispersion obtained: Yes.
- Log Kow of the test chemical: Not relevant. The test system does not have log Kow limitations. [Log Kow of the test item: 5.6, (Anon., OECD TG 117, 2020)]. Further enhancements can be made to increase solubility for ‘difficult test items’. Through choice of 1-step dilution direct into test medium and/or alternative test system compatible solvents. See “Other:” below.
- Other: GARDskin assay does not have the limitations of other OECD TG 442E methods (such as h-CLAT). See other comments below.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 500 μM (in-well concentration)
- Solubility in solvents: Solubility testing was performed to identify the most appropriate vehicle for the test item. The maximum assessed concentration in vehicle was 500 mM in DMSO. Under the test method: the test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a test system compatible vehicle to a minimum in-well concentration of 1 μM. For the test item, the maximum target in-well concentration of any test item was defined to be 500 μM. If the test item was not soluble to the maximum in-well concentration of 500 μM, the vehicle that generated the highest test item in-well concentration is utilised, with a minimum recommended in-well concentration of 1 μM. Based on solubility testing, the vehicle chosen for downstream procedures was DMSO (0.1%) and the highest soluble in well concentration was determined to 500 μM.
- Solubility in incubation medium: No issues reported.
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: Cytotoxicity testing was performed between 500 μM and 1 μM in-well concentration. The results of the cytotoxicity assessment indicated cytotoxicity (see table 1). The Rv90 was shown to be between 50 μM and 100 μM in-well concentration. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item. Relative Viability (%) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%.
- Final concentration range selected on basis of: Not applicable. “For cytotoxic test items, the concentration yielding 90% relative viability (Rv90) should be used for the GARD assay. For non-cytotoxic test items, a concentration of 500 µM should be used if possible. For non-toxic compounds that are insoluble at 500 µM in cell media, the highest soluble concentration should be utilised. Whichever of these three criteria is met, only one concentration will be used for the genomic assay. The concentration to be used for any given chemical is termed the ‘GARD input concentration’ (sic)”. Reference: H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169.
- Other: Additional notes applicable to ‘difficult test items’:
The applicant is aware of an acceptable standard protocol deviation that may be used to enhance the sensitivity of the test assay for difficult test items which show solubility limitations and show little to no cytotoxicity. Therein, the vehicle diluted test Items are directly diluted (1-step dilution) in the cell medium in order to assess the highest (potential) GARD assay "in-well concentration". The cell medium diluted test item is further transferred to the SenzaCells for stimulation according to the Standard GARD Assay Protocol.
Basis:
(i) Where the test Item shows solubility limitations in cell medium when using the Standard GARD Assay Protocol, a 1-step dilution in vehicle may be used to increase the in-well concentration to the upper limit of the titration range.
(ii) Where no cellular cytotoxicity is observed, the GARD input concentration for the test item is determined to 500 µM. Input concentration would only be limited by cytotoxicity considerations.
References:
(1) H. Johansson et al., The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27 (2013) 1163–1169
(2) Test system manufacturer: [Senzagen AB, Sweden] published research (2019) indicates: DMSO (0.1%), water and/or Acetone, DMF, DMF/Glycerol, Ethanol, Glycerol, Isopropanol have been determined all compatible solvents with the test system, the latter of which are used increase its applicability domain (by increasing test item solubility) when testing difficult to dissolve test items: Applicant assessment indicates these solvent options are likely to be incorporated in future OECD TG publication, and/or have been demonstrated by the manufacturer to be compatible with the test system.

This standard protocol deviation would be used to enhance the sensitivity of the test assay by maximising concentration/exposure of the test item to the SenzaCell : human myeloid leukaemia-derived cell line by utilising the highest soluble concentration and/or 500 µM concentration, where possible in the absence of cytotoxicity and a determinable Rv90 (relative viability 90%).These dilution and solvent options are likely to be incorporated in future OECD TG publication, and/or have been demonstrated by the manufacturer to be compatible with the test system.

Since the test item was fully soluble to 500 μM in DMSO (0.1%) this protocol deviation was not required in the present study. A standard vehicle, DMSO (0.1%) was also utilised.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: Triplcate (3)
- Number of repetitions: Single (1)
- Test chemical concentrations: 500 μM (in-well concentration) = ‘GARD input concentration’
- Application procedure: In the main experiment(s) the cells are stimulated (as previously described) using the GARD input concentration. In addition to the assayed test Item a set of concurrent positive and negative controls are performed as reference and quality controls. Test item and controls were assayed in biological replicates (n=3). After incubation for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity, cell culture was lysed in TRizol reagent and stored at -20 ± 5 °C until RNA was extracted/isolated. In parallel, stimulated cells were PI stained and analysed by flow cytometry to verify the expected relative viability. RNA isolation from lysed cells was performed using commercially available kits (referenced in the full study report). Total RNA was quantified, and quality controlled using BioAnalyzer equipment.
- Exposure time: incubation for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity (then cell culture lysis)
- Study evaluation and decision criteria used: Endpoint measurement:
(i) Gene expression analysis using Nanostring nCounter System : A total of 100 ng of RNA was used as sample input in a hybridization assay with the GPS (GARD Prediction Signature) specific Reporter CodeSet. The hybridized sample was prepared on chip using nCounter Preparation Station and individual transcripts of the GPS were quantified using Digital Analyzer. Data was imported into relevant software environment for statistical computation analysis (documented in the full study report). Raw data was normalised using a gene specific Counts Per Total Counts (CPTC) algorithm.
(ii) Prediction model and evaluation: For assessment of skin sensitisation, the Support Vector Machine (SVM) was modelled on a training data set corresponding to samples used for assay development. For a comprehensive overview of the training data set and methods, this is referenced in the full study report. Batch variations between the training data set and the test data set were eliminated using the Batch Adjustment by Reference Alignment (BARA) method, using unstimulated cells as a reference control. Each sample in the test set was assigned a Decision Value IDV) based on its transcriptional levels of the endpoint specific biomarker signature.

EVALUATION CRITERIA:
WITHIN GARDSKIN:
Each sample in the test set were assigned a Support Vector Machine (SVM) Decision Value (DV), based on its transcriptional levels of the GPS biomarker signature.
Test item considered ‘negative’ where: mean DV < 0 (n ≥ 2) which results in no classification as a skin sensitiser (or ‘non-sensitising’).
Test item considered ‘positive’ where: mean DV ≥ 0 (n ≥ 2) which results in classification as a skin sensitiser.
WITHIN GARDPOTENCY:
For potency classification, a test item assigned a median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A). Consequently, a test item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B).
Further information on GARDpotency sub-model is provided in ‘any other information on materials and methods incl. tables’.

- Description on study acceptance criteria: All applicable acceptability criteria were met.
(1) Positive Control: p-phenvlendiamine (PPD) [CAS 106-50-3] gave a positive prediction for sensitisation (passed GARDskin Assay Quality Control) and/or accurately classified.
(2) Negative Control: Dimethylformamide (DMF) [CAS 68-12-2 gave a negative prediction for sensitisation (passed GARDskin Assay Quality Control) and/or accurately classified.
(3) The test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a GARDskin and GARDpotency compatible vehicle to a minimum in-well concentration of 1 μM. (Unless standard protocol deviations utilised ; see above).
(4) The Main Stimulation cells should pass the Phenotypic Quality Control on the day of cell stimulation and the acceptance criteria for specified biomarker profile(s) for: CD86 (10-40%) ; CD54 (+ presence) ; HLA-DR (+ presence) ; CD80 (≤ 10%) ; CD34 (+ presence) ; CD14 (+ presence) ; CD1a (+ presence).
(5) The cell viability of the Main Stimulations should have fulfilled the following acceptance criteria: The positive control and a test item expected to induce cytotoxicity, should have a relative viability of 84.5 % - 95.4 %. The negative control and a test item not expected to induce cytotoxicity, should have a relative viability of ≥ 95.5 %. The unstimulated control should have an absolute viability of ≥ 84.5 %.
(6) For the RNA Quality Control : the total RNA concentration ≥ 20 ng/μL and/or a RIN value ≥ 8 and this should be observed in at least two test item replicates. For unstimulated (negative) controls and/or positive controls: individual replicates should pass.
(7) For the endpoint measurement quality control: the RCC files generated from the nCounter MAX analysis files should fulfil the following acceptance criteria: Imaging Quality: > 0.75 ; Linearity: > 0.95 ; Limit of Detection (LOD): (POS_E / LOD) > 1 and/or Binding Density (BDI: 0.05 < 80 < 2.25) and this should be observed in at least two test item replicates. For unstimulated (negative) controls and/or positive controls: individual replicates should pass.
- Other: No precipitation was reported observed at any point during the study including the start and end of the incubation period in solutions and wells.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): For cytotoxicity assessment, cells at passage number 4-16 were used. For Main Stimulation, cells at passage number 6-12 were utilised.
- Incubation conditions: For cytotoxicity assessment: 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity. For chemical stimulation of cells, exposed cells are incubated for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity.
- Washing conditions: After incubation for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2, the cells were lysed in TRlzol reagent and stored at -20 ± 5 °C awaiting RNA isolation. In parallel, cells from the same stimulation well were PI stained and analysed by flow cytometry as described in Cytotoxicity Assessment to verify the expected relative viability.
- Other: Details on and maintainece of the test cell line: The human myeloid leukaemia-derived cell line SenzaCell (available through ATCC), acting as an in vitro model of human Dendritic Cell (DC), is maintained in a-MEM supplemented with 20% (volume/volume) fetal calf serum and 40 ng/mL recombinant human Granulocyte Macrophage Colony Stimulating Factor (rhGM-CSF). During cell propagation, subculturing with fresh medium was performed every 2-3 days to a concentration of 0.2x10^6 cells/mL. Working stocks of cultures were grown for a maximum of 16 passages or two months after thawing.
Cells were seeded for stimulation directly following a cell split, i.e. the test item stimulations were scheduled to coincide with routine cell culture maintenance. The cell stimulations were initiated when a stable cell culture was established i.e. when at least a duplication of the cells between cell passages was seen, and, depending on the purpose of the cell stimulation, at specific cell passage ranges (see above).

DATA EVALUATION
- Cytotoxicity assessment: A test item that induces cytotoxicity was used at the concentration that induces 90 ± 5 % relative viability. This concentration ensures bioavailability of the test item, while not impairing immunological responses. A test item that was not cytotoxic (relative viability ca, 95.5%] was used at a concentration of 500 μM, or at the highest soluble concentration
- Prediction model used: Within GARDskin:
Each sample in the test set were assigned a Support Vector Machine (SVM) Decision Value (DV), based on its transcriptional levels of the GPS biomarker signature. Test item considered ‘negative’ where: mean DV < 0 (n ≥ 2) which results in no classification as a skin sensitiser (or ‘non-sensitising’). Test item considered ‘positive’ where: mean DV ≥ 0 (n ≥ 2) which results in classification as a skin sensitiser.
Within GARDpotency:
For potency classification, a test item assigned a median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A). Consequently, a test item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B).
Further information on GARDpotency sub-model is provided in ‘any other information on materials and methods incl. tables’.
- Other: Further information is provided in ‘any other information on results including tables’.

RESULTS:
For GARDSkin: the mean SVM DV was +2.02, the GARDskin Prediction was “sensitising”. All relevant concurrent positive and negative controls passed GARDskin Assay Quality Control criteria. All relevant validity criteria were met.
For GARDPotency: the median SVM DV was -0.915, the GARDpotency Prediction was: UN GHS Skin Sensitiser 1B since a test item assigned with median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A) and a test item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B).
Further information on GARDpotency sub-model is provided in ‘any other information on materials and methods incl. tables’ and/or ‘any other information on results including tables’.

Positive control results:

Positive Control: p-phenvlendiamine (PPD) [CAS 106-50-3] gave a positive prediction for sensitisation (PC mean SVM Decision Value > 0 (actual: +8.21) and/or median SVM Decision Value = > 0 (actual: +4.35 ; indicating a PC UN GHS Skin Sensitiser 1A prediction) and passed GARDskin Assay and/or GARDpotency Quality Controls.

Key result

Run / experiment:

other: mean (n=3)

Parameter:

other: GARDSkin SVM Decision Value

Remarks:

TEST ITEM

Value:

2.02

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

mean SVM DV= -0.989 (non-sensitiser)

Positive controls validity:

valid

Remarks:

mean SVM DV = +8.21 (sensitiser)

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: median (n=3)

Parameter:

other: GARDPotency SMV Decision Value

Remarks:

TEST ITEM

Value:

-0.915

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

median SVM DV = +4.35 ; UN GHS Skin Sensitiser 1A prediction

Remarks on result:

other: median SVM DV = -0.915 ; UN GHS Skin Sensitiser 1B sub-category prediction under GARDpotency sub-model

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported. The results of the cytotoxicity assessment indicated cytotoxicity (see table 1). The Rv90 was shown to be between 50 μM and 100 μM in-well concentration. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item. Relative Viability (%) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The GARDskin main test assay was conducted in accordance with the GARDskin assay protocol ; non-exhaustive references include: OECD Test Guideline Program (TGP no. 4.106), and which has been EURL ECVAM validated according to test method: TM2011-09 (EU), and with reference to the non-exhaustive related publications: (1) H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381 ; and/or : (2) H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169. The test assay is pending full OECD acceptance. The laboratory where the study was conducted (details given in the full study report) achieved GLP certification: 26 May 2020 (inspected 06 to 15 April 2020). Information in the public domain. The study protocol was previously validated (in-house laboratory and/or external validations) with reference chemicals described in the published literature. The results of the testing on the reference chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). Additional public information on the in-house validation can be found in: H. Johansson et al. (2014), Genomic allergen rapid detection in-house validation--a proof of concept, Toxicol. Sci. ; 139 (2) : 362-70 and/or H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169 ; and/or for external validation within: H. Johansson et al., (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381. Additional information is in the public domain on the EURL ECVAM and OECD websites. The sub-test assay/sub-model: GARDpotency: is a related model – which has also been submitted for EURL ECVAM validated according to test method: TM2011-09 (EU) and/or is included in OECD Test Guideline Program (TGP no. 4.106). The main literature citation is : (4) R. Gradin et al. (2020) The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432. Further information on GARDpotency sub-model is provided in ‘any other information on materials and methods incl. tables’ and/or ‘any other information on results including tables’.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: No issues were reported in the full study report.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria: All applicable acceptability criteria were met. Further information is provided in ‘any other information on results including tables’.
(1) Positive Control: p-phenvlendiamine (PPD) [CAS 106-50-3] gave a positive prediction for sensitisation (passed GARDskin Assay Quality Control) and/or accurately classified.
(2) Negative Control: dimethyl sulfoxide (DMSO) [CAS 67-68-5] gave a negative prediction for sensitisation (passed GARDskin Assay Quality Control) and/or accurately classified.
(3) The test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a GARDskin and GARDpotency compatible vehicle to a minimum in-well concentration of 1 μM. (Unless standard protocol deviations utilised ; see above).
(4) The Main Stimulation cells should pass the Phenotypic Quality Control on the day of cell stimulation and the acceptance criteria for specified biomarker profile(s) for: CD86 (10-40%) ; CD54 (+ presence) ; HLA-DR (+ presence) ; CD80 (≤ 10%) ; CD34 (+ presence) ; CD14 (+ presence) ; CD1a (+ presence).
(5) The cell viability of the Main Stimulations should have fulfilled the following acceptance criteria: The positive control and a test item expected to induce cytotoxicity, should have a relative viability of 84.5 % - 95.4 %. The negative control and a test item not expected to induce cytotoxicity, should have a relative viability of ≥ 95.5 %. The unstimulated control should have an absolute viability of ≥ 84.5 %.
(6) For the RNA Quality Control : the total RNA concentration ≥ 20 ng/μL and/or a RIN value ≥ 8 and this should be observed in at least two test item replicates. For unstimulated (negative) controls and/or positive controls: individual replicates should pass.
(7) For the endpoint measurement quality control: the RCC files generated from the nCounter MAX analysis files should fulfil the following acceptance criteria: Imaging Quality: > 0.75 ; Linearity: > 0.95 ; Limit of Detection (LOD): (POS_E / LOD) > 1 and/or Binding Density (BDI: 0.05 < 80 < 2.25) and this should be observed in at least two test item replicates. For unstimulated (negative) controls and/or positive controls: individual replicates should pass.
Further information on GARDpotency sub-model is provided in ‘any other information on materials and methods incl. tables’ and/or ‘any other information on results including tables’.

Table 1. Test item cytotoxicity assessment:

	Relative viability (%)
In-well concentration (µM)	100	90	80	70	60	50
Test item #1 replicate	90.20	85.35	91.04	93.52	95.80	95.54
Test item #2 replicate	87.92	91.67	92.97	96.36	95.52	97.55
Test item #3 replicate	89.79	91.26	92.11	94.23	95.82	96.75

 

Table 2. Relative viability of Test and Reference Items and the result of the RNA Quality Controls .

Test/Reference Item	Replicate	Stimulation conc (i)	Vehicle	Relative viability (ii) (%)	Acceptance criteria (Pass/Fail)	RNA conc (ng/µL)	RIN	RNA QC (Pass/Fail)
Test Item	1	100 µM	DMSO	90.20	Pass	22	9.8	Pass
	2	100 µM	DMSO	87.92	Pass	22	10.0	Pass
	3	100 µM	DMSO	89.79	Pass	50	10.0	Pass
Positive control (PPD)	1	75 µM	DMSO	87.66	Pass	74	9.8	Pass
	2	75 µM	DMSO	88.13	Pass	48	9.7	Pass
	3	75 µM	DMSO	91.21	Pass	69	10.0	Pass
Negative control (DMSO)	1	0.1 %	Not applicable	100.05	Pass	39	9.9	Pass
	2	0.1 %	Not applicable	99.43	Pass	23	10.0	Pass
	3	0.1 %	Not applicable	100.10	Pass	22	9.8	Pass
Unstimulated control (absolute viability %)	1	Not applicable	Not applicable	96.50 (iii)	Pass	28	10.0	Pass
	2	Not applicable	Not applicable	96.05 (iii)	Pass	36	10.0	Pass
	3	Not applicable	Not applicable	97.00 (iii)	Pass	40	9.9	Pass

(i) Concentration based on max. solubility and cytotoxicity.

(ii) Observed viability, calculated based on the equation in section Standard GARDskin Dose-Response Assay Procedure.

(iii) Absolute Viability

 

Table 3. Result of the Endpoint measurement Quality Control for the GARDskin assessment.

Test/Reference Item	Replicate	Imaging Quality	Binding Density	Limit of detection	Linearity	Endpoint measurement QC (Pass/Fail)
Test item	1	0.95	0.49	11.35	0.999	Pass
	2	0.96	0.46	8.81	0.998	Pass
	3	0.96	0.50	13.48	0.999	Pass
Positive control (PPD)	1	0.96	0.45	9.078	0.997	Pass
	2	0.95	0.48	12.03	0.998	Pass
	3	0.96	0.48	12.92	0.998	Pass
Negative control (DMSO)	1	0.94	0.45	11.54	0.999	Pass
	2	0.95	0.48	14.08	0.999	Pass
	3	0.96	0.38	10.99	0.996	Pass
Unstimulated control	1	0.93	0.51	11.63	0.998	Pass
	2	0.94	0.45	11.60	0.998	Pass
	3	0.96	0.43	10.68	0.996	Pass

 

Table 4. Result of the Endpoint measurement Quality Control for the GARDpotency assessment.

Test/Reference Item	Replicate	Imaging Quality	Binding Density	Limit of detection	Linearity	Endpoint measurement QC (Pass/Fail)
Test item	1	0.97	0.29	4.83	0.998	Pass
	2	0.97	0.28	5.59	0.999	Pass
	3	0.98	0.30	4.66	0.997	Pass
Positive control (PPD)	1	0.97	0.23	4.95	0.997	Pass
	2	0.98	0.26	4.67	0.998	Pass
	3	0.97	0.27	4.85	0.998	Pass
Unstimulated control	1	0.98	0.30	4.66	0.997	Pass
	2	0.97	0.28	5.20	0.996	Pass
	3	0.94	0.24	4.61	0.998	Pass

 

Table 5.Table12: Decision Values and GARDskin classifications.

Test/Reference Item	Decision value (DV)			Mean DV	GARDskin classification	Acceptance criteria (i) (Pass/Fail)
	Replicate #1	Replicate #2	Replicate #3
Test item	+1.32	+2.75	+2.01	+2.02	Sensitizer	Not applicable for Test Item
Positive control (PPD)	+7.88	+7.82	+8.94	+8.21	Sensitizer	Pass
Negative control (DMSO)	-1.18	-1.37	-0.419	-0.989	Non-sensitizer	Pass

(i) Reference Item acceptance criteria as described in section Standard GARD Assay Acceptance Criteria.

 

Table 6: Decision Values and GARDpotency classifications.

Test/Reference Item	Decision value (DV)			Median DV	GARDpotency classification	Acceptance criteria (i) (Pass/Fail)
	Replicate #1	Replicate #2	Replicate #3
Test item	-0.265	-0.954	-0.915	-0.915	UN GHS SS 1B	Not applicable for Test Item
Positive control (PPD)	+3.70	+4.35	+4.53	+4.35	UN GHS SS 1A	Pass

(i) Reference Item acceptance criteria as described in section Standard GARD Assay Acceptance Criteria.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the condition of this study, the test item is considered to be sensitising to the skin. The test item gave a mean Support Vector Machine (SVM) Decision Value (DV) of +2.02. The GARDskin Prediction was sensitising since the mean SVM DV > 0 (n ≥ 2). In the GARDpotency sub-model the test item gave a median SVM DV was -0.915 and is predicted to be weak sensitiser (UN GHS Skin Sensitiser 1B). All relevant concurrent positive and negative controls passed GARDskin and GARDPotency Assay Quality Control criteria. All applicable acceptability criteria were met.

Executive summary:

The study was performed to the GARDskin and GARDpotency (Genomic Allergen Rapid Detection) test methods and/or models which has been EURL ECVAM validated [test method: TM2011-09 (EU)] and is included in the OECD Test Guideline Program (TGP no. 4.106), pending full OECD TG adoption. The test assay is detailed in the following non-exhaustive related publications: (1) H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381 ; and/or : (2) H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169 ; and/or (3) A.Forreryd et. al. (2016), From genome-wide arrays to tailor-made biomarker readout – Progress towards routine analysis of skin sensitizing chemicals with GARD, Toxicology in Vitro 37, 178–188 and/or (4) R. Gradin et al. (2020) The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432. The GARD platform is a versatile testing strategy that relies on gene expression analysis of cell cultures exposed to test items in vitro. Information on the GARDpotency prediction model – training set is provided in: R. Gradin et al. (2020). The finalised prediction model for potency sub classification was based upon: Model 2; SVM based on 51 genomic biomarkers and Genomic Allergen Rapid Detection input concentration as listed in Gradin et al. (2020). The ‘GARD input concentration’ is based upon dose-dependent cytotoxicity measurements. Batch variations between the training data set and the test data set were eliminated using the Batch Adjustment by Reference Alignment (BARA) methodology, see: Gradin et al., (2019) Batch adjustment by reference alignment (BARA): Improved prediction performance in biological test sets with batch effects, PLoSONE 14(2): e0212669. Each sample in the test set was assigned a Decision Value (DV) based on its transcriptional levels of the endpoint specific biomarker signature. For potency classification, a test item assigned a median SVM DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A). Consequently, a test item assigned a median SVM DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B). Within the present study, prior to the assay the test item was assessed for solubility and cytotoxic effect in order to establish the GARD input concentration (concentration inducing 90% relative viability). Cytotoxicity testing was performed between 500 μM and 1 μM in-well concentration. Solubility testing was performed to identify the most appropriate vehicle for the test item. The maximum assessed concentration in vehicle was 500 mM in DMSO. Under the test method: the test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a test system compatible vehicle to a minimum in-well concentration of 1 μM. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item. Relative Viability (%) (mean ; n=3 replicates) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%. In the main experiment(s) the cells are stimulated (as previously described) using the GARD input concentration. The concurrent positive control was p-phenvlendiamine (PPD) [CAS 106-50-3], for which an in-well concentration of 75 µM in DMSO was utilised. The concurrent negative control was dimethyl sulfoxide (DMSO) [CAS 67-68-5], for which an in-well concentration of 0.10% was utilised. Under the condition of this study, within the GARDskin assay the test item is considered to be sensitising to the skin. The test item gave a mean Support Vector Machine (SVM) Decision Value (DV) of +2.02. The GARDskin prediction was sensitising since the mean SVM DV > 0 (n ≥ 2). Under the GARDpotency sub-model, the median SVM DV was -0.915 and since a test item assigned a median DV < 0, the test item is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B). The positive and negative controls gave acceptable results, respectively. The phenotypic quality control, the cytotoxicity/relative viability quality controls, the RNA quality control and the endpoint measurement quality controls all met the relevant acceptance criteria. All applicable acceptability criteria were considered to be met.

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) No. 1907/2006 Annex XI, section 1.2 – weight of evidence, justification for not performing further skin sensitisation in vivo studies includes expert assessment: The category: salicylates, were reviewed by Belsito et al., (2007). A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients. Food and Chemical Toxicology, 45, S318–S361. Therein the salicylates, with the exception of benzyl salicylate, were considered based on all the available data to have ‘some potential for skin sensitization’ but that in general they possess either ‘no or very limited skin sensitization potential’ in humans. As such the conclusion was that for the category in general: it could be inferred that members therein were either non-classified or GHS Skin Sensitisation category 1B (i.e. low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed). This correlates with the conclusion of the GARDpotency (2021) assay, conducted on the substance using a method consistent with OECD Test Guideline Program (TGP no. 4.106) and/or EURL ECVAM test method: TM2011-09 (EU). Additional references on the test method are provided in R. Gradin et al. (2020). The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432. The available data indicates a weight of evidence that the substance is a skin sensitiser and/or should be classified within GHS Skin Sensitisation category 1B. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.3, July 2017) further testing is not scientifically justified.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin Sensitisation:

1. Key Study – Molecular initiating Key Event 1: DPRA, OECD TG 442C, 2020 : The study was performed according to the OECD TG 442C in chemico Direct Peptide Reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and 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. The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the SPCC depletion for the test item, were all within the acceptability criteria for the DPRA. the standard deviation value of the SPCL depletion was above the acceptability criterium for the DPRA as stated in the OECD 442C guideline. As this was caused by the occurrence of a phase separation in the test item samples in combination with interference of a test item related peak with SPCL, DPRA results were accepted. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed in the test item samples. An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion were presented. In the cysteine reactivity assay the test item showed 1.1% SPCC depletion while in the lysine reactivity assay the test item showed 21.4% SPCL depletion (estimated value). The mean of the SPCC and SPCL depletion was 11.3% (estimated value) and as a result the test item was considered to be positive in the DPRA. As the overlap in retention time between the test item and SPCL was incomplete, the SPCL depletion was estimated and used in the Cysteine 1:10 / Lysine 1:50 prediction model, however, assignment to a reactivity class was not made.

 

Applicant assessment indicates: the test item gave a positive result in the DPRA and was not classified into a reactivity class (formally, within the study) due to phase separation being observed in the incubation period in SPCC and SPCL test item-samples.

However applicant assessment indicates using the Cysteine 1:10 / Lysine 1:50 prediction model :

(i) the Mean of SPCC and SPCL depletion was in the > 6.38 < 22.62 - predicted low reactivity (Positive) class

(ii) the estimated result for lysine is suggested as possible: > 22.62 < 42.47 - predicted moderate reactivity (Positive) class

The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

 

2. Key Study – Molecular initiating Key Event 2: KeratinoSens, OECD TG 442D, 2020 : The study was performed to the OECD TG 442D in vitro Skin Sensitisation guideline: ARE-Nrf2 Luciferase Test Method under GLP. The objective of this study was to evaluate the ability of the test item, to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in two independent experiments. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. The test item precipitated at dose levels of 250 μM and above at the start and end of the incubation period, except in the second experiment, where precipitation at the start of the incubation period was only observed at 500 μM. The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between two standard deviations of the historical mean (97 µM and 56 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in experiment 2 (2.08-fold and 3.56-fold in experiment 1 and 2, respectively). It was noted for the second experiment, the concentration of 31 μM already showed a luminesce induction of 1.56. However, since the concentration of 63 μM showed an induction of 1.48, the EC1.5 was calculated from the point were a clear dose related induction was observed. The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 9.0% in experiment 1 and 2, respectively). In these experiments, the cells were incubated with the test item in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) or 0.24 – 500 µM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. The test item showed toxicity (IC30 values of 37 μM and 41 μM and IC50 values of 45 μM and 48 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.29-fold and 1.32-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM. All relevant test acceptability criteria were met.

 

3(a). Key study – Molecular initiating Key Event 3: GARDSkin Assay, 2021 : The study was performed to the GARDskin (Genomic Allergen Rapid Detection) test method which has been EURL ECVAM validated [test method: TM2011-09 (EU)] and is included in the OECD Test Guideline Program (TGP no. 4.106), pending full OECD TG adoption. The test assay is detailed in the following non-exhaustive related publications: (1) H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381 ; and/or : (2) H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169 ; and/or (3) A. Forreryd et. al. (2016), From genome-wide arrays to tailor-made biomarker readout – Progress towards routine analysis of skin sensitizing chemicals with GARD, Toxicology in Vitro 37, 178–188 and/or (4) R. Gradin et al. (2020) The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432. The GARD platform is a versatile testing strategy that relies on gene expression analysis of cell cultures exposed to test items in vitro. The high-dimensional readout allows for gene expression analysis of customized biomarker signatures, each specific for various biological end points. In addition, the final prediction gives rise to the classification of test items based on a statistical prediction model that is in turn dependent on an end point specific training data set as to if a substance is skin sensitising or non-skin sensitising. The human myeloid leukaemia-derived cell line SenzaCell (available through ATCC), acting as an in vitro model of human Dendritic Cell (DC), is maintained in a-MEM supplemented with 20% (volume/volume) fetal calf serum and 40 ng/mL recombinant human Granulocyte Macrophage Colony Stimulating Factor (rhGM-CSF). During cell propagation, subculturing with fresh medium was performed every 2-3 days to a concentration of 0.2x10^6 cells/mL. Working stocks of cultures were grown for a maximum of 16 passages or two months after thawing. Cells were seeded for stimulation directly following a cell split, i.e. the test item stimulations were scheduled to coincide with routine cell culture maintenance. The cell stimulations were initiated when a stable cell culture was established i.e. when at least a duplication of the cells between cell passages was seen, and, depending on the purpose of the cell stimulation, at specific cell passage ranges. For cytotoxicity assessment, cells at passage number 4-16 were used. For Main Stimulation, cells at passage number 6-12 were utilised. To verify that cells were maintained in an inactivated state, and to detect potential phenotypic drift, the cells were stained with a panel of biomarkers, CD54, CD86, HLA-DR, CD34, CDS0, CD14 and CD1a. For chemical stimulation of cells, exposed cells are incubated for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity. Prior to the assay: the test item was assessed for solubility and cytotoxic effect in order to establish the GARD input concentration (concentration inducing 90% relative viability). Cytotoxicity testing was performed between 500 μM and 1 μM in-well concentration. Solubility testing was performed to identify the most appropriate vehicle for the test item. The maximum assessed concentration in vehicle was 500 mM in DMSO. Under the test method: the test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a test system compatible vehicle to a minimum in-well concentration of 1 μM. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item. Relative Viability (%) (mean ; n=3 replicates) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%. In the main experiment(s) the cells are stimulated (as previously described) using the GARD input concentration. In addition to the assayed test Item a set of concurrent positive and negative controls are performed as reference and quality controls. Test item and controls were assayed in biological replicates (n=3). After incubation for 24 ± 0.5 h at 37 ± 1 °C, 5 ± 0.5 % CO2 and 95% humidity, cell culture was lysed in TRizol reagent and stored at -20 ± 5 °C until RNA was extracted/isolated. In parallel, stimulated cells were PI stained and analysed by flow cytometry to verify the expected relative viability. RNA isolation from lysed cells was performed using commercially available kits (referenced in the full study report). Total RNA was quantified, and quality controlled using BioAnalyzer equipment. The test item indicated no cellular cytotoxicity, therefore the GARD input concentration was determined to 100 µM for the test item. The concurrent positive control was p-phenvlendiamine (PPD) [CAS 106-50-3], for which an in-well concentration of 75 µM in DMSO was utilised. The concurrent negative control was dimethyl sulfoxide (DMSO) [CAS 67-68-5], for which an in-well concentration of 0.10% was utilised. Under the condition of this study, the test item is considered to be sensitising to the skin. The test item gave a mean Support Vector Machine (SVM) Decision Value (DV) of +2.02. The GARDskin prediction was sensitising since the mean SVM DV > 0 (n ≥ 2). Under the separate GARDpotency sub-model, the prediction was UN GHS Skin Sensitiser 1B as the median SVM DV was -0.915 since a test item assigned with median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A) and a test item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B). Further information on GARDpotency sub-model is provided separately. The positive and negative controls gave acceptable results. The phenotypic quality control, the cytotoxicity/relative viability quality controls, the RNA quality control and the endpoint measurement quality controls all met the acceptance criteria. All applicable acceptability criteria were considered to be met.

 

3(b). Key study – POTENCY : GARDPotency Assay, 2021 : The study was performed to the GARDskin and GARDpotency (Genomic Allergen Rapid Detection) test methods and/or models which has been EURL ECVAM validated [test method: TM2011-09 (EU)] and is included in the OECD Test Guideline Program (TGP no. 4.106), pending full OECD TG adoption. The test assay is detailed in the following non-exhaustive related publications: (1) H. Johansson et al. (2019), Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitisers: Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 170, 374-381 ; and/or : (2) H. Johansson et al. (2013), The GARD assay for assessment of chemical skin sensitizers, Toxicology in Vitro 27, 1163–1169 ; and/or (3) A.Forreryd et. al. (2016), From genome-wide arrays to tailor-made biomarker readout – Progress towards routine analysis of skin sensitizing chemicals with GARD, Toxicology in Vitro 37, 178–188 and/or (4) R. Gradin et al. (2020) The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432. The GARD platform is a versatile testing strategy that relies on gene expression analysis of cell cultures exposed to test items in vitro. Information on the GARDpotency prediction model – training set is provided in: R. Gradin et al. (2020). The finalised prediction model for potency sub classification was based upon: Model 2; SVM based on 51 genomic biomarkers and Genomic Allergen Rapid Detection input concentration as listed in Gradin et al. (2020). The ‘GARD input concentration’ is based upon dose-dependent cytotoxicity measurements. Batch variations between the training data set and the test data set were eliminated using the Batch Adjustment by Reference Alignment (BARA) methodology, see: Gradin et al., (2019) Batch adjustment by reference alignment (BARA): Improved prediction performance in biological test sets with batch effects, PLoSONE 14(2): e0212669. Each sample in the test set was assigned a Decision Value (DV) based on its transcriptional levels of the endpoint specific biomarker signature. For potency classification, a test item assigned a median SVM DV ≥ 0 is classified as a strong skin sensitizer (UN GHS Skin Sensitiser 1A). Consequently, a test item assigned a median SVM DV < 0 is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B). Within the present study, prior to the assay the test item was assessed for solubility and cytotoxic effect in order to establish the GARD input concentration (concentration inducing 90% relative viability). Cytotoxicity testing was performed between 500 μM and 1 μM in-well concentration. Solubility testing was performed to identify the most appropriate vehicle for the test item. The maximum assessed concentration in vehicle was 500 mM in DMSO. Under the test method: the test item should be soluble (e.g. no noticeable precipitation or phase separation) by ocular inspection, in a test system compatible vehicle to a minimum in-well concentration of 1 μM. Since the test item indicated cellular cytotoxicity, the GARD input concentration was determined to be 100 µM for the test item. Relative Viability (%) (mean ; n=3 replicates) : At 50 µM: ca. 96.61% ; 60 µM: ca. 95.7% ; 70 µM: ca. 94.7% ; 80 µM: ca. 92.0% ; 90 µM: ca. 89.4% ; and 100 µM: ca. 89.3%. In the main experiment(s) the cells are stimulated (as previously described) using the GARD input concentration. The concurrent positive control was p-phenvlendiamine (PPD) [CAS 106-50-3], for which an in-well concentration of 75 µM in DMSO was utilised. The concurrent negative control was dimethyl sulfoxide (DMSO) [CAS 67-68-5], for which an in-well concentration of 0.10% was utilised. Under the condition of this study, within the GARDskin assay the test item is considered to be sensitising to the skin. The test item gave a mean Support Vector Machine (SVM) Decision Value (DV) of +2.02. The GARDskin prediction was sensitising since the mean SVM DV > 0 (n ≥ 2). Under the GARDpotency sub-model, the median SVM DV was -0.915 and since a test item assigned a median DV < 0, the test item is classified as a weak skin sensitizer (UN GHS Skin Sensitiser 1B). The positive and negative controls gave acceptable results, respectively. The phenotypic quality control, the cytotoxicity/relative viability quality controls, the RNA quality control and the endpoint measurement quality controls all met the relevant acceptance criteria. All applicable acceptability criteria were considered to be met.

 

4. Expert Judgement: In accordance with REACH Regulation (EC) No. 1907/2006 Annex XI, section 1.2 – weight of evidence, justification for not performing further skin sensitisation in vivo studies includes expert assessment: The category: salicylates, were reviewed by Belsito et al., (2007). A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients. Food and Chemical Toxicology, 45, S318–S361. Therein the salicylates, with the exception of benzyl salicylate, were considered based on all the available data to have ‘some potential for skin sensitization’ but that in general they possess either ‘no or very limited skin sensitization potential’ in humans. As such the conclusion was that for the category in general: it could be inferred that members therein were either non-classified or GHS Skin Sensitisation category 1B (i.e. low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed). This correlates with the conclusion of the GARDpotency (2021) assay, conducted on the substance using a method consistent with OECD Test Guideline Program (TGP no. 4.106) and/or EURL ECVAM test method: TM2011-09 (EU). Additional references on the test method are provided in R. Gradin et al. (2020). The GARDpotency Assay for Potency-Associated Subclassification of Chemical Skin Sensitizers—Rationale, Method Development, and Ring Trial Results of Predictive Performance and Reproducibility, Toxicol. Sci. 176, 423–432. The available data indicates a weight of evidence that the substance is a skin sensitiser and/or should be classified within GHS Skin Sensitisation category 1B. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.3, July 2017) further testing is not scientifically justified.

 

Weight of Evidence Conclusion:

The applicant assesses by expert judgement the available information. Within the battery of in chemico and in vitro test assays, there is negative to positive predictions for sensitisation. The test item appears to be either a non-sensitiser to weak sensitiser. Using a precautionary principle conclusion through evaluation of all the available information, the substance is considered a weak sensitiser and to have a low potency (e.g. EC3 >> 2%) based on the weight of evidence. The substance is assigned to GHS Classification: Skin Sensitisation Category 1B. Further information concerning the weight of evidence conclusion is attached by the applicant in attachment to this endpoint. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation: category 1B.

 

Within the battery of in chemico and in vitro test assays, there is are equivocal to positive predictions for sensitisation. The test item appears to be either a non-sensitiser to weak sensitiser. Using a precautionary principle conclusion through evaluation of all the available information, the substance is considered a weak sensitiser and to have a low potency (e.g. EC3 >> 2%) based on the weight of evidence. The 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.

References:

(1) ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, July 2017)

(2) OECD (2017) Guidance Document on the Reporting of Defined Approaches and Individual Information Sources to be Used within Integrated Approaches to Testing and Assessment (IATA) for Skin Sensitisation. OECD Series on Testing and Assessment No. 256