[No public or meaningful name is available]

Endpoint summary

Description of key information

Weight of Evidence, skin sensitising 1B, 2021: 

1. Key Event 1: negative but uncertain due to observation of precipitate and phase separation after the incubation period for both SPCC and SPCL (or minimal peptide reactivity class), DPRA, OECD TG 442C, 2019

2. Key Event 2: negative (no LC induction > 1.5 in 2 of 2 experiments at up to 2000 µM) (n=3 replicates), KeratinoSens, OECD TG 442D, 2019

3. Key Event 3: UN GHS category 1B skin sensitiser, : skin sensitizer (mean SMV Decision Value (DV) of +4.12, and since SMV DV >0 and category 1B since according to GARDpotency SMV DV= -1.54 (DV<0). Genomic Allergen Rapid Detection (GARD): GARDskin and GARDpotency assays, 2021

 

Conclusion: 

Weight of evidence conclusion: 

The applicant assesses by expert judgement the available information. Within the battery of in vitro test assays, there are equivocal, negative and 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 based on the weight of evidence. 

The substance is assigned to 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:

28-10-2020 until 16-12-2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Precipitation and phase separation occurred after the incubation period for both Cysteine and Lysine samples. Phase separation was also observed upon preparation of both samples.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
1) Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.2 mg of SPCC in 20.36 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication. 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.

2) Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.0 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes. 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.

- Preparation of the test chemical 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 (MeOH) and ethanol (EtOH).
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. For the SPCC assay, a 20-fold dilution was prepared by mixing one volume of the test item stock solution with fifteen volumes of phosphate buffer pH 7.5 and four volumes of ACN. For the SPCL assay, a 4-fold dilution was prepared by mixing one volume of the test item stock solution with three volumes of ammonium acetate buffer pH 10.2. The presence of cloudiness, precipitate and/or phase separation was evaluated by visual inspection to aid solvent selection for the main study.
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assays 37.33 mg of test item was pre-weighed into
a clean amber glass vial and dissolved, just before use, in 1792 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the 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. Any residual volumes were discarded.

- Preparation of the positive controls, reference controls and co-elution controls:
(i) Positive Controls:
Three replicates of Positive Control (PCcys-1 to PCcys-3) were prepared by mixing 750 μL of the Phosphate buffer pH 7.5 solution, 200 μL of the ACN solution and 50 μL of the Cinnamic aldehyde solution (100mM in ACN).
Three replicates of Positive Control (PClys-1 to PClys-3) were prepared by mixing 750 μL of the Stock solution of 0.667 mM SPCL and 250 μL of the Cinnamic aldehyde solution (100mM in ACN).
(ii) Reference controls:
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.
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 SPCL was subsequently calibrated at multiple concentrations.
(iii) Co-elution controls: Co-elution control (CC) solution (CCcys) was prepared by mixing 750 μL of the Phosphate buffer pH 7.5 with 200 μL of the ACN solution and 50 μL of test item test solution (100 mM). Co-elution control (CC) solution (CClys) was prepared by mixing 750 μL of the Ammonium acetate buffer pH 10.2 with 250 μL of the test item test solution (100 mM).

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 24.1 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours.

- Precipitation noted: Prior to HPLC analysis the samples were visually inspected for precipitation. The test item samples that showed precipitation and/or phase separation were centrifuged (at 400 g) for 5 minutes at room temperature and supernatant was transferred to a new vial.
Upon preparation of the SPCC and SPCL test item samples, phase separation was observed.
After incubation of the SPCC and SPCL test item samples, both a precipitate and phase
separation was observed.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys
1) SPCC calibration curve:
The SPCC standard calibration curve is presented in the full study report. The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
2) SPCL calibration curve:
The SPCL standard calibration curve is presented in the full study report. The correlation coefficient (r2) of the SPCL standard calibration curve was 1.0000. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.

- Verification of the suitability of the HPLC for test chemical and control substances:
(i) Acceptability of the Cysteine reactivity assay: The mean peptide concentration of Reference Controls A was 0.499 ± 0.0003 mM and the mean peptide concentration of Reference Controls C was 0.496 ± 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
ndicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
(ii) Acceptability of the Lysine reactivity assay:
The mean peptide concentration of Reference Controls A was 0.501 ± 0.001 mM and the mean peptide concentration of Reference Controls C was 0.501 ± 0.003 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.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0.2 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
Precipitation and phase separation occurred after the incubation period for both Cysteine and
Lysine samples. Phase separation was also observed upon preparation of both samples.

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for reference controls A to C: All criteria met.
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Table1: Acceptability of the DPRA

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.9998	>0.99	1.0000
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.499 ± 0.0003	0.50 ± 0.05	0.501 ± 0.001
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.496 ± 0.002	0.50 ± 0.05	0.501 ± 0.003
CV (%) for RC samples B and C	<15.0	0.7	<15.0	0.7
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	71.6	40.2-69.0	62.9
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.4	<11.6	0.2
SD of peptide depletion for the test item (%)	<14.9	0.4	<11.6	0.0

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

 

Table 2: Results of the DPRA with the test item

	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test Item	0.2%	±0.4 %	0.0%	±0.0%	0.1%	Negative: No or minimal reactivity

Interpretation of results:

GHS criteria not met

Remarks:

Test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since a precipitate and phase separation was observed after the incubation period for both SPCC and SPCL, and phase separation was also observed upon preparation of both samples, one cannot be sure how much test item remained in the solution to react with the peptides. This negative result is uncertain and should be interpreted with due care. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.

Conclusions:

The test item gave a negative in DPRA and was classified in the "Negative: No or minimal reactivity class" using the Cysteine 1:10 / Lysine 1:50 prediction model. The result will be considered within a weight of evidence assessment for Classification and Labelling purposes.

Executive summary:

The objective of this study was to determine the reactivity of Test item towards 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 spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which assigns the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD 442C guideline.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

All the assay validation criteria were met. 

 

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 peptide depletion for the test item, were all within the acceptability criteria for the DPRA as stated in the OECD 442C guideline.

Upon preparation of the SPCC and SPCL test item samples, phase separation was observed. After incubation of the SPCC and SPCL test item samples, both a precipitate and phase separation was observed.

In the cysteine reactivity assay the test item showed 0.2% ±0.4 % SPCC depletion while in the lysine reactivity assay the test item showed 0.0% ±0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.1 % and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

 

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. Test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since a precipitate and phase separation was observed after the incubation period for both SPCC and SPCL, and also phase separation was also observed upon preparation of both samples, one cannot be sure how much test item remained in the solution to react with the peptides. This negative result is uncertain and should be interpreted with due care. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30-10-2020 until 23-02-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution:
A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless solution).

- Preparation of the test chemical serial dilutions:
In the main experiments the test item was dissolved in DMSO at 200 mM (clear colourless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solutions 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 (final concentration DMSO of 1%) in the first and second experiment. All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.
No precipitation was observed at the start and end of the incubation period in the 96-well plates.

- Preparation of the positive controls:
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in the full study report, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

- Preparation of the solvent, vehicle and negative controls:
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

SOLUBILITY ASSESSMENT:

- Highest concentration used:
The 100-fold dilution of the 200 mM DMSO stock showed slight precipitation. This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).

- Solubility in solvents:
In order to obtain a suitable solvent, a solubility test was performed prior to testing based on visual assessment. The solvents in order of preference were, DMSO (dimethyl sulfoxide), Milli-Q water and ethanol. The test item was dissolved in DMSO (clear colourless solution).

- Solubility in incubation medium:
The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in the final test concentrations . All formulations formed a clear solution.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates:
All concentrations of the test item were tested in triplicate. 3 replicates for each positive control, 18 wells per plate for the vehicle control, 3 blank wells per plate (no cells, no treatment)

- Number of repetitions:
2 experiments were performed: Experiment 1 and Experiment 2.

- Test chemical concentrations:
2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration DMSO of 1%) in the first and second experiment.

- Application procedure:
The medium is 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 chemical
and control substances are added.

- Exposure time:
48 hours ± 1 h

- Study evaluation and decision criteria used:
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met
in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is
considered negative (Figure 1):
1. The Imax is equal or 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 ≥ 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.

- Description on study acceptance criteria:
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene
dimethacrylate glycol, should be statistically significant equal or above the threshold
of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) 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. If the latter criterion is not fulfilled, the dose-response of Ethylene
dimethacrylate glycol should be carefully checked, and tests may be accepted only if
there is a clear dose-response with increasing luciferase activity induction at
increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the
vehicle (negative) control DMSO should be below 20% in each repetition which
consists of 18 wells tested. If the variability is higher, results should be discarded.
If the variability is higher, a maximum of three of the eighteen wells may be excluded
based on the Dixon’s Q-test. If the variability is still higher, the results should be
discarded.
All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.


SEEDING AND INCUBATION:
- Seeding conditions (passage number and seeding density):
For testing, cells were 80-90% confluent. The passage number used was P+24 in experiment 1 and P+28 in experiment 2. The passage number used in experiment 2 was P+28, instead of the maximum of P+25 described in the OECD Guideline. This study plan deviation did not affect the integrity of the study because both the vehicle control and positive control passed the predetermined acceptance criteria.

- Incubation conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions
were a humid atmosphere of 80 - 100% (actual range 52 – 93%), containing 5.0 ± 0.5% CO2
in air in the dark at 37.0 ± 1.0°C (actual range 35.2 – 37.0°C). Temperature and humidity
were continuously monitored throughout the experiment. The CO2 percentage was monitored
once on each working day. Temporary deviations from the temperature and humidity
occurred due to opening and closing of the incubator door. Based on laboratory historical data
these deviations are considered not to affect the study integrity.

- Precipitation noted:
No precipitation was observed at the start and end of the incubation period in the 96-well plates.

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test:
Luminometer: TECAN Infinite® M200 Pro Plate Reader.

- Plate used: 96 wells.

- Lysate preparation:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate
(lyophilized) from Promega (Leiden, The Netherlands) 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).

DATA EVALUATION
- Cytotoxicity assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure
time with fresh DMEM Glutamax containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1;
Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C
± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were
lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each
well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200
Pro Plate Reader.

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

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

other: EDMG (Ethylene dimethacrylate glycol)

Positive control results:

• 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 within two standard deviations of the historical mean (65 µM and 54 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.74-fold and 2.57-fold in experiment 1 and 2, respectively).

• The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 6.2% in experiment 1 and 2, respectively).

Historical results for luciferase induction by the positive control in the test laboratory: average and standard deviations from 503 valid runs are presented in the full study report.

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.22

Cell viability:

> 70%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.27

Cell viability:

> 70%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No EC1.5 > 1.5 could be determined; no luciferase activity induction.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Cell viability:

> 70%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No EC1.5 > 1.5 could be determined; no luciferase activity induction.

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

Both tests (Experiment 1 and Experiment 2) passed the acceptance criteria:
• 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 within two standard deviations of the historical
mean (65 μM and 54 μM in experiment 1 and 2, respectively). A dose response was
observed and the induction at 250 μM was higher than 2-fold (2.74-fold and 2.57-fold in
experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle
(negative) control DMSO was below 20% (6.5% and 6.2% in experiment 1 and 2,
respectively).
Overall it is concluded that the test conditions were adequate and that the test system
functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed toxicity (IC30 values of 74 μM and 85 μM and IC50 values of 88 μM
and 98 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 1.27-fold and 1.22-fold in
experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM
assay since negative results (<1.5-fold induction) were observed at test concentrations up to
2000 μM.

Executive summary:

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.
The study procedures described in this report were based on the most recent OECD TG 442D guideline (25 June 2018).
The test item was dissolved in dimethyl sulfoxide (DMSO) 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) in the first and second experiment. The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.
Both tests passed the acceptance criteria. 
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
The test item showed toxicity (IC30 values of 74 μM and 85 μM and IC50 values of 88 μM and 98 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 1.27-fold and 1.22-fold in experiment 1 and 2 respectively.

The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

In conclusion, the Test Item is classified as negative (no activation of the
antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Reference 3

Endpoint:

skin sensitisation: in vitro

Remarks:

Genomic Allergen Rapid Detection method

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08-03-2021 to 16-04-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Genomic Allergen Rapid Detection (GARDTM) is an in vitro assay designed to predict the ability of chemical substances to induce skin sensitisation based on the analysis of the relative expression levels of a biomarker signature of 196 genes using a human myeloid leukemia cell line called SenzaCell. The GARDTM is based on chemical stimulation of the SenzaCell line, acting as an in vitro model of human Dendritic Cells (DCs). The readout of the assay is a transcriptional quantification of the genomic predictors, collectively termed the GARDTM Prediction Signature (GPS), using Nanostring nCounter technology. 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 (2012) and OECD 255 (2016) and OECD 256 (2016). GARDskin is included in the OECD Test Guideline Program (TGP 4.106) and currently undergoing ESAC peer-review.

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

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 the OECD Test Guideline Program (TGP no. 4.106)

Deviations:

no

Principles of method if other than guideline:

- Principle of test:
The Genomic Allergen Rapid Detection (GARD) methods are a series of assays for hazard assessment characterization and risk assessment of sensitizers.
The test method is based on the nCounter system and measures the expression level of a panel of genes in the human myeloid cell line MUTZ-3 exposed to chemicals. In GARDskin, the expression of a panel of 200 genes (the GARD Prediction Signature, GPS) is used as input to a prediction model based on a Support Vector Machine (SVM) trained with 38 chemicals. This model classifies chemicals as skin sensitising or non-sensitising. The GARDpotency can be considered as second-tier of a testing strategy, following GARDskin. Thus, in GARDpotency, the model sub-categorises sensitisers into GHS/CLP categories 1A (strong/extreme sensitisers) or 1B (weak/moderate sensitisers) based on the expression of a panel of 52 genes (the GARD Potency Prediction Signature, GPPS).

- Short description of test conditions:
In the study, Test Item was soluble in DMSO at an in-well concentration of 500 µM (0.1% in-well concentration of DMSO). In the cytotoxicity assessment cells were incubated with different concentrations (500 µM - 1 µM) of the Test Item. After exposure, cells were stained with propidium iodide and cell viability was measured by FACS analysis. Cytotoxicity was observed, and 150 µM was the concentration that induced a relative viability within the acceptance criteria and therefore chosen for further downstream processing. Following cellular stimulations RNA was isolated and endpoint measurements were performed using the GARDskin and GARDpotency genomic prediction signatures. All samples passed the standard acceptance criteria for the GARDskin and GARDpotency assays.

- Parameters analysed / observed:
Once the cytotoxicity was determined for the Test Item, Main stimulations were performed with individual preparations of the Test Item, Reference Items (negative control and positive control) and unstimulated control. Individual cell cultures were used to achieve three biological replicate cell samples of each Test Item, Reference Item, and unstimulated control which passed the acceptance criteria for Main stimulation as stated in the section Relative Viability Quality Control. After incubation for 24±0.5 h at 37±1 °C and 5±0.5 % CO2, the cells were lysed in TRIzol reagent (Life Technologies) and stored at -20±5 °C awaiting RNA isolation. In parallel, cells from the same stimulation well were PI stained and analyzed by flow cytometry as described in Cytotoxicity Assessment to verify the expected relative viability.
After that, RNA was isolated from the TRIzol-lysed cells according to manufacturer’s instructions (Zymo-Research). An RNA sample aliquot was taken from each RNA sample for RNA quantification and quality control. Both aliquots and RNA samples were stored at -80 °C.
For RNA quantification, the RNA sample aliquots were thawed on ice and assayed with the Bioanalyzer 2100 according to manufacturer’s instructions. For each completed RNA analysis, a software generated PDF report was saved together with raw XAD files. The PDF report contains software calculated values for total RNA content and RNA Integrity Number (RIN) for each analyzed sample. All RNA samples were verified against the RNA acceptance criteria. Samples that passed the criteria were used for the Endpoint Measurements.
A total of 100 ng of RNA was used as sample input in a hybridization assay with the endpoint specific GARDskin and GARDpotency CodeSet (NanoString Technologies, Seattle, WA). Each hybridized sample was prepared on cartridge using nCounter MAX Prep Station and individual transcripts of the endpoint specific biomarker signature were quantified using nCounter MAX Digital Analyzer (NanoString Technologies, Seattle, WA).
Raw data files, Reporter Code Count (RCC) files, were exported from the nCounter MAX Digital Analyzer and quality controlled, according to the section Standard GARD Assay Acceptance Criteria below, using the GARD Data Analysis Application (GDAA) for classification of skin sensitizers and CLP classification. Data files that do not pass the Acceptance Criteria were removed from the classification analysis.
For assessment of skin sensitization hazard and potency properties, a Support Vector Machine previously modelled on a training data set corresponding to samples used for assay development was used. Each sample in the test set was assigned a Decision Value (DV) based on its transcriptional levels of the endpoint specific biomarker signature.
For the GARDskin binary hazard classification of skin sensitizers, a Test Item or control assigned a mean DV ≥ 0 is classified as a skin sensitizer (UN GHS category 1). Consequently, a Test Item or control assigned a mean DV < 0 is classified as a skin non-sensitizer (UN GHS No category).

For potency classification, a Test Item assigned a median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS category 1A). Consequently, a Test Item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS category 1B).

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Activation of dendritic cells

Details on the study design:

Skin sensitization (in vitro test system)
- The study was conducted in accordance with the GARDSkin and GARDpotency 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 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.

- A solubility test was performed. 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+/- 5% relative viability).

- Details on and manteinance of the test cell line: The Test system consists of the human myeloid leukemia-derived cell line SenzaCell™ acting as an in vitro model of a human Dendritic Cell (DC) and is maintained in α-MEM medium (Cytiva) supplemented with 20 % (volume/volume) fetal calf serum (Life Technologies, Carlsbad, CA) and 40 ng/ml recombinant human Granulocyte Macrophage Colony Stimulating Factor (rhGM-CSF, Miltenyi Biotec, Germany). During cell propagation, subculturing with fresh medium was performed every 2-3 days to a concentration of 0.2×106 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 used.
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, CD80, CD14 and CD1a.

- Solubility assessment: The Test Item solubility was assessed: The vehicle chosen for downstream procedures was DMSO and the highest soluble in well concentration was determined to be 500 µM. The Test Item was freely soluble at the maximum in well-concentration of 500 µM, and no solubility issues were reported.

- Positive Control preparation and concentrations: The positive control p-phenvlendiamine (PPD) [CAS 106-50-3] of purity ≥ 99% (source and batch information in the full study report), for which an in-well concentration of 75 µM in DMSO was utilised. Positive controls were concurrently tested in a manner similar to test item described below.

- Negative Control preparation and concentrations: The negative control Dimethyl sulfoxide (DMSO) [CAS 67-68-5] of purity ≥ 99.5% (source and batch information in the full study report), for which an in-well concentration of 0.10% was utilised. Negative controls were concurrently tested in a manner similar to test item described below.

- Test Item preparation and concentrations: test item was dissolved in DMSO and the highest soluble in well concentration was determined to 500 µM. For test item dissolved in DMSO, the in-well concentration of DMSO was 0.1%. Cytotoxic effects of the test item were monitored. After incubation for 24 h at 37±1 °C and 5±0.5 % CO2, harvested cells were stained with the viability marker Propidium Iodide (PI) (BD Bioscience, USA) and analyzed by flow cytometry. PI-negative cells were defined as viable, and the relative viability (Rv) of cells stimulated with each concentration in the titration range was calculated. A Test Item that induces cytotoxicity was used at the concentration that induces 90±5 % relative viability (Rv90). This concentration ensures bioavailability of the Test Item, while not impairing immunological responses. A Test Item that was not cytotoxic (relative viability ≥95.5%) was used at a concentration of 500 µM, or at the highest soluble concentration. The solubility and cytotoxicity assessment were used as guidance to find the appropriate stimulation concentration during Main Stimulation.

- Main stimulation: In the main experiment the cells are stimulated 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 three biological replicates performed at different time-points and using different cell cultures. After incubation for 24±0.5 h at 37±1 °C and 5±0.5 % CO2, the cells were lysed in TRIzol reagent (Life Technologies) and stored at -20±5 °C awaiting RNA isolation. 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 (BioAnalyzer 2100) equipment.

- 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 endpoint specific GARDskin and GARDpotency CodeSet (NanoString Technologies, Seattle, WA). Each hybridized sample was prepared on cartridge using nCounter MAX Prep Station and individual transcripts of the endpoint specific biomarker signature were quantified using nCounter MAX Digital Analyzer (NanoString Technologies, Seattle, WA).
Raw data files, Reporter Code Count (RCC) files, were exported from the nCounter MAX Digital Analyzer and quality controlled, according to the section Standard GARD Assay Acceptance Criteria below, using the GARD Data Analysis Application (GDAA) for classification of skin sensitizers and CLP classification. Data files that do not pass the Acceptance Criteria were removed from the classification analysis.
(ii) Prediction model and evaluation: For assessment of skin sensitization hazard and potency properties, a Support Vector Machine previously modelled on a training data set corresponding to samples used for assay development was used. For a comprehensive overview of the training data set and methods, see Forreryd et al., 2016 and Zeller et al., 2017 (for further details on biomarker discovery, see Johansson et al., 2011). Batch variations between the training data set and the test data set were eliminated using the Batch Adjustment by Reference Alignment (BARA) method (Gradin et al., 2019), using unstimulated cells as 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: For assessment of skin sensitization hazard and potency properties, a Support Vector Machine previously modelled on a training data set corresponding to samples used for assay development was used. For a comprehensive overview of the training data set and methods, see Forreryd et al., 2016 and Zeller et al., 2017 (for further details on biomarker discovery, see Johansson et al., 2011). Batch variations between the training data set and the test data set were eliminated using the Batch Adjustment by Reference Alignment (BARA) method (Gradin et al., 2019), using unstimulated cells as 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.
For the GARDskin binary hazard classification of skin sensitizers, a Test Item or control assigned a mean DV ≥ 0 is classified as a skin sensitizer (UN GHS category 1). Consequently, a Test Item or control assigned a mean DV < 0 is classified as a skin non-sensitizer (UN GHS No category).

For potency classification, a Test Item assigned a median DV ≥ 0 is classified as a strong skin sensitizer (UN GHS category 1A). Consequently, a Test Item assigned a median DV < 0 is classified as a weak skin sensitizer (UN GHS category 1B).

- 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.

- Acceptability 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)
(2) Negative Control: Dimethyl sulfoxide (DMSO) [CAS 67-68-5 gave a negative prediction for sensitisation (passed GARDSkin Assay Quality Control).

- RESULTS:
The mean SMV DV was +4.12, the GARDSkin Prediction was sensitising.
The mean SMV DV was -1.54 for the GARDpotency Prediction it was classified as 1B.
All relevant concurrent positive and negative controls passed GARDSkin Assay Quality Control criteria. All relevant validity criteria were met.

Positive control results:

Positive Control: p-phenvlendiamine (PPD) [CAS 106-50-3] gave a positive prediction for sensitisation (PC SMV Decision Value > 0) and passed GARDSkin Assay Quality Control. It also gave a positive prediction for sensitisation (1A) and passed GARDpotency Assay Quality Control.

Key result

Run / experiment:

other: mean (n=3)

Parameter:

other: GARDskin SMV Decision Value

Value:

4.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

See vehicle controls (served as negative control)

Positive controls validity:

valid

Remarks on result:

other: Indication of skin sensitisation

Key result

Run / experiment:

other: mean (n=3)

Parameter:

other: GARDpotency SMV Decision Value

Value:

-1.54

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

See vehicle controls (served as negative control)

Positive controls validity:

valid

Remarks on result:

other: Indication of skin sensitisation further subcategorized as UN GHS category 1B in the GARDpotency assay, when tested at an in-well concentration of 150 µM.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported. No (cyto)toxicity was observed during testing with the test item to the cell line.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study was conducted in accordance with the GARDSkin and GARDpotency assays protocols ; 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.

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.
(1) Positive Control: p-phenvlendiamine (PPD) [CAS 106-50-3] gave a positive prediction for sensitisation (passed GARDSkin and GARDpotency Assay Quality Control)
(2) Negative Control: Dimethyl Sulfoxide (DMSO) [CAS 67-68-5 gave a negative prediction for sensitisation (passed GARDSkin Assay Quality Control)
No precipitation was reported observed at any point during the study including the start and end of the incubation period in solutions and wells.

Interpretation of results:

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

Remarks:

The test item gave a positive result in the GARDskin test based on the mean SMV DC > 0 (test conducted in triplicate). The test test item gave a negative result in the GARDpotency test based on the mean SMV DC < 0 (test conducted in triplicate). The result will be used within a weight of evidence assessment for C&L purposes.

Conclusions:

Under conditions of this study, the test item us considered to be skin sensitiser to the skin categorized 1B. The test item gave a mean Support Vector Machine (SVM) Decision Value (DV) of +4.12 in the GARDskin test and a SVM DV of -1.54 in the GARDpotency test.
In conclusion, based upon the results of this Study, the test item was classified as a sensitizer in the GARDskin assay and was further subcategorized as UN GHS category 1B in the GARDpotency assay, when tested at an in-well concentration of 150 µM.

Executive summary:

Based on the results of this Study, the test item was classified as a sensitizer in the GARDskin assay and was further subcategorized as UN GHS category 1B skin sensitizer in the GARDpotency assay, when tested at an in-well concentration of 150 µM.

 

The GARDskin assay provides hazard classification of skin sensitizers. The method evaluates the transcriptional patterns of an endpoint-specific genomic biomarker signature, referred to as the GARDskin Genomic Prediction Signature (GPS), in the SenzaCell™ cell line exposed to a Test Item. The high-dimensional transcriptional data is analyzed by classification algorithms, which provide binary classification of Test Items as sensitizers or non-sensitizers. The GARDskin assay has been demonstrated to be transferable, reproducible, and functional, with an estimated accumulated predictive accuracy of 94 %, well balanced between sensitivity (92.7 %) and specificity (96.0 %) (Johansson et al., 2019).

The GARD™potency test method is used to discriminate between UN GHS sub-category 1A skin sensitizers and a combination of sub-category 1B and non-sensitizers according to UN GHS classification criteria. The GARDpotency assay was validated in an inter-laboratory ring trial (Gradin et al., 2020), conducted in adherence with OECD guidance documents for acceptance of novel alternative methods. The method was demonstrated to be transferable, reproducible, and functional, with an estimated accumulated predictive accuracy of 88 %, across the participating laboratories.

 

In this study, the test item was soluble in DMSO at an in-well concentration of 500 µM (0.1 % in-well concentration of DMSO). In the cytotoxicity assessment cells were incubated with different concentrations (500 µM - 1 µM) of the Test Item. After exposure, cells were stained with propidium iodide and cell viability was measured by FACS analysis. Cytotoxicity was observed, and 150 µM was the concentration that induced a relative viability within the acceptance criteria and therefore chosen for further downstream processing. Following cellular stimulations RNA was isolated and endpoint measurements were performed using the GARDskin and GARDpotency genomic prediction signatures. All samples passed the standard acceptance criteria for the GARDskin and GARDpotency assays.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

1.- Key study - Molecular initiating Key Event 1: DPRA, OECD TG 442C, 2020: NEGATIVE, unconclusive.

2.- Key study - Molecular initiating Key Event 2: Keratinosens, OECD TG 442D, 2021: NEGATIVE. 

3.- Key study - Molecular initiating Key Event 3: GARDskin and GARDpotency assays, 2021: POSITIVE, Skin sensitiser category 1B.

 

Weight of evidence conclusion: 

The applicant assesses by expert judgement the available information. Within the battery of in vitro test assays, there are equivocal, negative and 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 based on the weight of evidence. 

The substance is assigned to GHS classification: Skin Sensitisation Category 1B. 

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 as skin sensitiser category 1B.