Reaction mass of 1-[rac-(1R,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol isomer 1 and 1-[rac-(1S,6S)-2,2,6-trimethylcyclohexyl]pentan-3-ol

Endpoint summary

Description of key information

In the DPRA assay, the obtained results are inconclusive for dendritic cells activation (due to precipitation).

In the KeratinoSens assay, the test item showed no skin sensitizing potential based on the key event "activation of keratinocytes".

In the U-Sens assay, the test item showed skin sensitizing potential based on the key event "activation of dendritic cells".

The negative results of the in vitro tests were confirmed with QSAR estimations using DEREK Nexus v6.0 and the Danish Q(SAR) Database.

In conclusion, the test item is not classified as skin sensitiser.

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:

from 2018-01-03 to 2018-02-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptides:
Cysteine- (SPCC) containing peptide: Ac-RFAACAA-COOH (MW=750.9 g/mol)
Lysine- (SPCL) containing peptide: Ac-RFAAKAA-COOH (MW=775.9 g/mol)

Material and technical equipment:
- System 1 (used for Cysteine Reactivity Assay)
Surveyor MS HPLC pump
MPS 3C autosampler
LC Column oven 300
Surveyor PDA detector
- System 2 (used for Lysine Reactivity Assay)
Surveyor MS HPLC pump
HTC PAL autosampler
Column Oven #151006
Surveyor PDA detector

Controls:
For Cysteine Reactivity Assay:
Positive control (PC): Cinnamic aldehyde, prepared as 100 mM in ACN, SPCC stock solution and ACN
Co-elution control: Phosphate buffer pH 7.5, ACN and the test substance without peptide.
For Lysine Reactivity Assay:
Positive control (PC): Cinnamic aldehyde, prepared as 100 mM in ACN, and SPCL stock solution
Co-elution control: Ammonium acetate buffer pH 10.2 and the test substance without peptide.

Test substance preparation:
33.21 mg and 35.25 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1563 µL and 1659 µL ACN, respectively, to obtain 78.2 mM solutions. No correction for the purity/composition of the test item was performed.
Vehicle: Acetonitrile, ACN
Reason for the vehicle: A solubility test to assesse the test items solubility in different solvents was performed before the main test. ACN was chosed as the best solvent in which the test item dissolved and did not for precipitation or cloudy solution.

Test Item Samples:
- Test item solution, ACN and SPCC stock solution
- Test item and SPCL stock solution

Preparation of peptide stock solutions:
SPCC - A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication. 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.
SPCL - A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 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.

Experimental procedure:
After preparation, the samples were placed in the autosampler in the dark and incubated at 25 ± 2.5 °C. The incubation period was 24.5 hours for RCcysB-samples and 23 hours for the RClysB-samples. All samples were analysed with HPLC-PDA analysis. Prior to HPLC PDA analysis the samples were visually inspected for precipitation.

Key result

Parameter:

other: mean % SPCC depletion

Value:

0.46

Positive controls validity:

valid

Remarks:

78.3 %

Key result

Parameter:

other: mean % SPCL depletion

Value:

0

Positive controls validity:

valid

Remarks:

52.8 %

Key result

Parameter:

other: mean % depletion

Value:

0.23

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
Formation of precipitation of the test item by addition of peptide solution.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.

In the cysteine reactivity assay the test item showed 0.46 % SPCC depletion while in the lysine reactivity assay the test item showed 0.0 % SPCL depletion. The mean of the SPCC and SPCL depletion was 0.23 % and as a result the 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. Therefore, the test item was considered to be negative in the DPRA.

Since no correction was used to prepare the test item stock solution in ACN, the prepared test item stock solution had a concentration of 78.2 mM instead of the desired 100 mM and the SPCC and SPCL incubations were performed at lower concentrations than intended. However, since at this lower concentration precipitation was already observed upon addition of the test item to the SPCC and SPCL peptide solutions, not applying a correction factor to correct for the purity (78.2 %) of the test item had no impact on the outcome of the study.

Table 1. SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for 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	0.46 %	±0.43 %	0.0 %	±0.0 %	0.23 %	Negative: No or minimal reactivity

SD = Standard Deviation

Table 2. SPCC Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCC Depletion
PCcys-1	567923	0.112	77.0 %
PCcys-2	568110	0.112	77.0 %
PCcys-3	476032	0.093	80.7 %
		Mean	78.3 %
		SD	2.1 %

Table 3. SPCC Peak Area, Concentration, Depletion and Area Ratio (A₂₂₀/A₂₅₈) of the Test Item Samples

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCC Depletion	Peak area at 258 nm (µAU)	Area ratio (A220/A258)
209239/A-cys-1	2450576	0.502	0.8 %	124186	19.73
209239/A-cys-2	2457863	0.504	0.5 %	126457	19.44
209239/A-cys-3	2483781	0.509	0.0 %	128952	19.26
		Mean	0.46 %	NA	19.48
		SD	0.43%	NA	0.24

NA = Not applicable

Table 4. SPCL Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCL Depletion
PClys-1	1108440	0.234	51.1 %
PClys-2	1055792	0.222	53.4 %
PClys-3	1049112	0.221	53.7 %
		Mean	52.8 %
		SD	1.4 %

Table 5. SPCL Peak Area, Concentration and Depletion and Area Ratio (A₂₂₀/A₂₅₈) of the Test Item Samples

Sample code	Peak area at 220 nm (µAU)	Concentration (mM)	SPCL Depletion	Peak area at 258 nm (µAU)	Area ratio (A220/A258)
209239/A-lys-1	2347019	0.513	0.0 %	146582	16.01
209239/A-lys-2	2277900	0.497	0.0 %	145013	15.71
209239/A-lys-3	2302332	0.503	0.0 %	146599	15.70
		Mean	0.0 %	NA	15.81
		SD	0.0 %	NA	0.18

Interpretation of results:

other: inconclusive for dendritic cells activation (due to precipitation)

Conclusions:

In conclusion, the 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 precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, the result is regarded as 'inconclusive for dendritic cells activation (due to precipitation)'.

Executive summary:

A study according to OECD TG 442C - Direct Peptide Reactivity Assay (DPRA) was conducted under GLP to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with SPCC or SPCL for 24 hours at 25 °C, the relative peptide concentration was determined by HPLC-PDA detection at 220 and 258 nm. As reference control cinnamic aldehyde was used. The test item was tested at test concentration of 78.2 mM and the reference item at 100 mM. As a vehicle acetonitrile (ACN) was used. Synthetic peptide control solutions of SPCC (containing Ac-RFAACAA-COOH) and SPCL (containing Ac-RFAAKAA-COOH) were prepared in ACN. Co-eluent samples containig only the test item and the respective buffer were also tested under the same conditions. After the incubation period samples were injected directly into the HPL-PDA system. The concentration of SPCC or SPCL was photometrically determined at 220 and 258 nm. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. Following this model, the mean % SPCC depletion of the test item was 0.46 % and the mean % SPCL depletion was 0 %. The average % depletion of the test item was determined to be 0.23 %. The reference control showed mean % SPCC depletion of 78.3 % and a mean % SPCL depletion of 52.8 %. All validity criteria were met and the test is considered as valid. As a precipitation was observed after adding the peptide solutions to the test item, the result is regarded as 'inconclusive for dendritic cells activation (due to precipitation)'.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-02-12 to 2018-04-24

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

Type of study:

activation of keratinocytes

Details on the study design:

- Vehicle: dimethyl sulfoxide (DMSO)
- Solvent/vehicle control (VC): yes, 1 % DMSO in exposure medium. Eighteen wells were tested per plate.
- Positive control (PC): Ethylene dimethacrylate glycol, CAS 50-21-5
- Blank control: on each plate three blank wells were tested (no cells, no treatment).

Test substance preparation:
A correction of 1.28 was made for the purity of the test item (sum of isomers 78.2 %). A solubility test in DMSO was perfomed prior to testing based on visual assessment. The test item stock solution was prepared in DMSO to a final concentration of 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series): 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20 and 0.098 mM. The stock solution and the spike solutions were diluted 25-fold with exposure medium (final concentration DMSO of 4 %). These solutions were diluted 4-fold in the assay resulting to final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98 μM. All concentrations were tested in triplicates. Two independent assay runs were performed.
Precipitation was observed at the start of the incubation period at test concentrations of 250 µM and upwards and at the end of the incubation period at test concentrations of 63 µM and upwards.

Positiv control preparation:
A 2-fold dilution series ranging from 0.78 to 25 mM was prepared in DMSO and was diluted following the same procedure as for the test item to a final concentration of 7.8 to 250 μM (final concentration of DMSO of 1 %). All concentration were tested in triplicates in each assay run.

Test system:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element was used (e.g. the KeratinoSens™ cell line). Upon receipt, cells were propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.

Cell culture:
Basic medium - Dulbecco’s minimal supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum.
Maintenance medium - Dulbecco’s minimal supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum and geneticin (500 μg/mL).
Exposure medium - Dulbecco’s minimal supplemented with 1 % (v/v) heat-inactivated (56 °C; 30 min) fetal calf serum.

Environmental conditions:
All incubations were carried out in a humid atmosphere (80 - 100 %) containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.5 - 36.4 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Experimental procedure:
- Subculturing: Cells were subcultured upon reaching 80 - 90 % confluency.
- Plating of Cells: One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. One plate was 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+16 in experiment 1 and P+18 in experiment 2.
- Treatment of Cells: 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 chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for 48 hours at 37 ± 1.0 °C in the presence of 5 % CO2. In total 2 experiments were performed.

Luciferase Activity Measurements:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega are mixed together. This substrate solution was kept at room temperature. The assay plates were removed from the incubator and the medium was 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 then shaken for at least 3 minutes at room temperature. Plates with the cell lysates were then placed in the luminometer to assess the quantity of luciferase (integration time at least 2 seconds).

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

Positive control results:

The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax in experiment 1 was 2.33 and the EC1.570 µM and in experiment 2 Imax was was 2.55 and the EC1.566 µM, respectively.

Key result

Run / experiment:

other: 1 and 2

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 1 and 2

Parameter:

other: I (max)

Value:

1.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no EC1.5 could be calculated

Run / experiment:

other: 1 and 2

Parameter:

other: IC30 in µM

Value:

35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 1 and 2

Parameter:

other: IC50 in µM

Value:

43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control,Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 
- The EC1.5of the positive control was between 5 and 125 µM (70 µM and 66 µM in experiment 1 and 2, respectively). A dose-response was observed and the induction at 250 µM was higher than 2-fold (2.33-fold and 2.55-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 % (7.9 % and 8.4 % in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Two independent experiments were performed. The cells were incubated with the test itemin a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours. 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.

Experiment 1

- Precipitation was observed at the start of the test at test concentration of 250 µM and upwards and at the end of the incubation period at test concentrations of 63 µM and upwards.

- The test item showed toxicity. The calculated IC30 was 39 µM and the calculated IC50 was 45 µM. 

- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.11 and, therefore, no EC1.5 could be calculated. 

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.33 and the EC1.5 70 µM. 

Experiment 2

- Precipitation was observed at the start of the test at test concentration of 250 µM and upwards and at the end of the incubation period at test concentrations of 63 µM and upwards. 

- The test item showed toxicity. The calculated IC30 was 35 µM and the calculated IC50 was 43 µM. 

- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.40 and, therefore, no EC1.5 could be calculated. 

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.55 and the EC1.5 66 µM.

Table 1. Overview Luminescence Induction and Cell Viability of the test item in Experiment 1 and 2

Concentration (μM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 luminescence	0.93	1.01	1.04	1.06	1.08	1.11	0.00*	0.00*	0.00*	0.00*	0.00*	0.00*
Exp 1 viability (%)	107.8	93.9	99.5	95.4	97.2	91.7	-0.2	-0.2	-0.1	-0.1	-0.1	0.0
Exp 2 luminescence	1.29	1.24	1.40	1.36	1.36	1.40	0.01*	0.00*	0.00*	0.00*	0.00*	0.00*
Exp 2 viability (%)	79.2	77.4	74.8	72.3	75.9	78.6	-0.1	-0.2	-0.1	0.3	0.0	0.1

* Precipitate observed at the end of the incubation period.

Table 2. Luminescence Induction of the test item in Experiment 1

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Measurement 1	0.94	1.01	1.00	1.02	1.08	1.10	0.00	0.00	0.00	0.00	0.00	0.00
Measurement 2	0.93	0.99	1.03	1.08	1.10	1.14	0.00	0.00	0.00	0.00	0.00	0.00
Measurement 3	0.93	1.02	1.08	1.08	1.07	1.09	0.00	0.00	0.00	0.00	0.00	0.00
Mean (fold-induction)	0.93	1.01	1.04	1.06	1.08	1.11	0.00	0.00	0.00	0.00	0.00	0.00

 

Table 3. Luminescence Induction of the test item in Experiment 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Measurement 1	1.27	1.25	1.36	1.33	1.29	1.49	0.00	0.00	0.00	0.00	0.00	0.00
Measurement 2	1.30	1.20	1.35	1.34	1.29	1.28	0.00	0.00	0.00	0.00	0.00	0.00
Measurement 3	1.29	1.28	1.49	1.41	1.49	1.44	0.02	0.00	0.00	0.00	0.00	0.00
Mean (fold-induction)	1.29	1.24	1.40	1.36	1.36	1.40	0.01	0.00	0.00	0.00	0.00	0.00

 

Table 4. Overview EC1.5, Imax, IC30 and IC50 Values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test item Experiment 1	NA	1.11	39	45
Test item Experiment 2	NA	1.40	35	43
Pos Control Experiment 1	70	2.33	NA	NA
Pos Control Experiment 2	66	2.55	NA	NA

NA = Not applicable

Interpretation of results:

other: no skin sensitizing potential based on the key event "activation of keratinocytes"

Conclusions:

The test item showed toxicity. No biologically relevant luciferase induction was measured at any of the test concentrations. 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 of this test up to the cytotoxicity limit.

Executive summary:

The in vitro KeratinoSens™ assay according to OECD 442D enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In the present study the test substance was dissolved in DMSO to a final concentration of 200 mM.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 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%).Cells were incubated with the test item for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction compared to the vehicle control. Under the condition of this study the test item is, therefore, considered as non sensitiser. The controls confirmed the validity of the study. The calculated EC1.5 was between 5 and 125 µM (70 µM in experiment 1; 66 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (7.9 % in experiment 1; 8.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 2.33 and in the second experiment it was 2.55, thus, the criteria were fulfilled. 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 of this test up to the cytotoxicity limit.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-02-06 to 2018-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E - Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)’

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

- Vehicle: 0.4 % DMSO in complete medium
- Vehicle control performed: yes
- Positiv control (PC): 2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599)
- Negative Control: Lactic Acid (LA, RS471)
- Blank control: on each plate three blank wells were tested (no cells, no treatment).

Test substance preparation:
A correction of 1.28 was made for the purity of the test item. A solubility test was perfomed prior to testing. The test item was dissolved at a concentration of 50 mg/mL in complete medium and DMSO. For the main experiment the test item was dissolved to a final concentration of 50 mg/mL in DMSO. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL and 200, 120, 70, 20, 15, 10 and 7.5 µg/mL in experiment 1 and 2, respectively, in the 96-well plate (final concentration DMSO of 0.4%). No precipitation was observed at the end of the incubation period in the 96-well plates. Test item concentrations were used within 3 hours after preparation.

Negative control preparation:
On the treatment day, a solution at 10 mg/mL of Lactic acid was prepared in RPMI medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution (final dose level 200 μg/mL).

Positiv control preparation:
2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599) was provided as 1 M solution. On the treatment day a 10 mg/mL solution was prepared in RPMI. This solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution (final dose level 50 μg/mL).

Test system: U937 human monocytes.
Justification: Inducible CD86-expressing cells
Stock cultures of these cells were stored in liquid nitrogen (-196 °C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. The cultures were checked for mycoplasma contamination.

Cell culture:
Cell culture medium - Stock and treatment cultures were performed in RPMI 1640 medium supplemented with 10 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively).

Environmental conditions:
All incubations were carried out in a humid atmosphere of 80 - 100 % (actual range 58 - 96 %) containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.8 - 36.4 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Experimental procedure:
Cultures were initiated in 96-well plates using 100 μL/well of a cell suspension adjusted at 5.0E+5 viable cells/mL.All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested. Cells were treated for 45 ± 3 hours with the selected doses. The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL. A untreated control (RPMI), vehicle control (DMSO) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL. In the second experiment cells were treated with seven selected doses of the test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 7.5, 10, 15 20, 70, 120 and 200 µg/mL.
Precipitation evaluation: After 45 ± 3 hours of exposure, wells were checked for precipitate. Two experiments were conducted.

Cell antibodies staining for IgG1 and CD86:
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200g). The supernatant was discarded and cells were rinsed once with Phosphate Buffered Saline (PBS) containing 5 % FCS. After a second centrifugation step, 100 μL/well of staining buffer (PBS containing 5 % FCS) was applied to the cells.
FITC-conjugated antibodies were used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control
- Human CD86 specific mouse IgG1
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 μL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 μL of PBS.

Flow cytometry method:
Acquisition:
Just before acquisition, 5 μL of a 0.5 μg/mL propidium iodide (PI) solution were added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition and the further analysis the BD FACSCanto™ flow cytometer was used.
Analysis:
All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted, if necessary, in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity was analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
Colour Interferences:
There was colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad was 50 % higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150 %).

Positive control results:

The positive control (TNBS) showed a S.I. ≥ 211% in two out of the three wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%) in experiment 1. Respectively, in experiment 2 the positive control (TNBS) showed a S.I. ≥ 291% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Key result

Run / experiment:

other: experiment 1 and 2

Parameter:

other: CV70 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: viability of cells was >70 %, therefore, CV70 could not be calculated and is considered >200 µg/mL

Key result

Run / experiment:

other: experiment 1

Parameter:

other: EC150 µg/mL (expression of CD86 cells)

Value:

11

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

S.I: <=101 %

Positive controls validity:

valid

Remarks:

S.I: >=211 %

Key result

Run / experiment:

other: experiment 2

Parameter:

other: EC150 µg/mL (expression of CD86 cells)

Value:

15

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

S.I: <=131 %

Positive controls validity:

valid

Remarks:

S.I: >=291 %

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90 % (97 % in experiment 1 and 100 % in experiment 2).
- The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90 % (98 % in experiment 1 and 100 % in experiment 2).
- The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250 % of the mean of the triplicate CD86 S.I. of untreated U937 cells in both experiments.
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2 % and ≤ 25 % in both experiments.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6 % and < 1.5 % in both experiments.
- No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.
In both experiments the positive and negative control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Two independent experiments were performed. The cell viability before incubation with the test item was > 90 % (96 % and 98 % in experiment 1 and 2, respectively). The cells were incubated withthe test itemin a concentration range of 1.0 – 200 µg/mL and 7.5 – 200 µg/mL in experiment 1 and 2, respectively. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide.

Experiment 1

- No precipitation was observed at the end of the incubation period in the 96-well plates.

- The test item showed no toxicity, the viability of the cells was higher than 70 % at all test concentrations and, therefore, no CV70 values could be calculated and is considered to be higher than 200 µg/mL.

- A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 11 µg/mL.

- The test item showed no colour interference.

-The positive control (TNBS) showed a S.I. ≥ 211 % in two out of the three wells and was non-cytotoxic at all concentrations (cell viability ≥ 70 %). The negative control (LA) showed a S.I. ≤ 101 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70 %).

Experiment 2

- No precipitation was observed at the end of the incubation period in the 96-well plates.

- The test item showed no toxicity, the viability of the cells was higher than 70 % at all test concentrations and, therefore, no CV70 values could be calculated and is considered to be higher than 200 µg/mL.

- A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 15 µg/mL.

- The test item showed no colour interference.

-The positive control (TNBS) showed a S.I. ≥ 291 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70 %). The negative control (LA) showed a S.I. ≤ 131 % in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70 %).

The test item showed no toxicity (CV70 value >200µg/mL). A biologically relevant induction of the CD86 activity (EC150 values of 11 µg/mL and 15 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test itemis considered for classification as Positive in the U-Sens™ assaysince positive results (>150 % increase) were observed at test concentrations with a cell viability of >70 % compared to the vehicle control.

Table 1. Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 ofthe test item

Test item Dose (µg/mL)	% Viability (Mean)*		CD86-IgG1 S.I.*		Colour Interference S.I.*
	Experiment		Experiment		Experiment
	1	2	1	2	1	2
1	97	-	104	-	107	-
7.5	-	100	-	108	-	102
10	96	99	139	124	116	110
15	-	99	-	148	-	103
20	78	93	217***	236***	95	100
50	89	-	185	-	101	-
70	-	99	-	188***	-	110
100	94	-	177***	-	119	-
120	-	99	-	156***	-	109
200	96	99	131	152***	122	104

* Values marked with *** are either below 70 % viability or above 150 S.I.

- Not Applicable

Table 2. Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the Positive, Negative and Vehicle Control

Controls	% Viability (Mean)*			CD86-IgG1 S.I.*
	Experiment			Experiment
	1	2		1	2
LA1	97	100		93	131
LA2	96	100		97	119
LA3	97	100		101	81
TNBS1	95	99		223***	358***
TNBS2	95	99		211***	371***
TNBS3	94	99		0	291***
DMSO	98	100		132	167
	IgG1 value (%)			CD86 basal expression (%)
	Experiment			Experiment
	1	2		1	2
RPMI1	0.6	0.7		16.2	12.0
RPMI2	1.3	1.3		15.5	12.1
RPMI3	1.2	1.4		16.4	10.0
RPMI Mean Viability				97	100
RPMI Drift				2 %	-22 %
LA Drift				7 %	-35 %

* Values marked with *** are either below 70 % viability or above 150 S.I.

 

Table3. Overview EC150 and CV70 Values

	EC150 (µg/mL)	CV70 (µg/mL)
Test item Experiment 1	11	NA
Test item Experiment 2	15	NA

NA = Not applicable

Interpretation of results:

other: some indication of skin sensitizing potential.

Conclusions:

In this study according to OECD 442E the test item activated CD86-IgG1 cells under the test conditions in at least three independent experiment runs. Therefore, the test item can be considered to have skin sensitizing potential based on the key event "activation of dendritic cells". However, the extreme bell-shaped dose-response curve casts doubts on the result reliability. The cell activation response at higher concentrations reverted back to vehicle levels in the absence of any cytotoxicity.

Executive summary:

This in vitro U937 cell line activation Test (U-Sens™) assay was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP). The test item was dissolved in DMSO at concentration of 50 mg/mL and administered to U937 cells for 45 ± 3 hours.In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL and 200, 120, 70, 20, 15, 10 and 7.5 µg/mL in experiment 1 and 2, respectively, in the 96-well plate (final concentration DMSO of 0.4 %).Positive and negative control ran parallel.FITC-conjugated antibodies were used for both IgG1 and CD86 staining. The test itemwas evaluated for the ability toincrease the expression levels of CD86 cell surface marker. No precipitation of the test item at the end of the experiments was observed. The test item showed no toxicity and the viability of the cells was higher than 70 % in all test concentrations, therefore, no CV70 values could be calculated andconsidered to be higher than 200 µg/mL. A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 for experiment 1 was 11 µg/mL and for experiment 2 was 15 µg/mL, respectively. All validity criteria were met and the test is considered as valid. Based on the obtained results from the two experiments, it can be considered that the test item has askin sensitizingpotentialbased on the key event "activation of dendritic cells". However, the extreme bell-shaped dose-response curve casts doubts on the result reliability. The cell activation response at higher concentrations reverted back to vehicle levels in the absence of any cytotoxicity.

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

Based on the SMILES code of the test item, the skin sensitising potential was estimated.

Qualifier:

according to guideline

Guideline:

other: Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals

Principles of method if other than guideline:

Based on the SMILES code of the test item, the skin sensitising potential was estimated.

GLP compliance:

no

Key result

Parameter:

other: skin sensitising potential

Value:

0

Remarks on result:

other: Skin sensitisation in mammals is negative. The substance is within the applicability domain of the model.

Interpretation of results:

other: Danish result: negative

Conclusions:

Using the Danish (Q)SAR Database, the skin sensitising potential of the test item was estimated to be negative. The substance is within the applicability domain of the model.

Executive summary:

The skin sensitising properties were estimated using Danish (Q)SAR Database. The skin sensitising potential of the test item was estimated to be negative. The test item is within the applicability domain.

Reference 5

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

An estimation of the skin sensitising properties of the test item using structural alert relationships was perfomed. The results of this estimation are used in a weight of evidence to support experimental in vitro results.

Qualifier:

according to guideline

Guideline:

other: Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals

Principles of method if other than guideline:

Estimates the skin sensitising properties of chemicals using structural alert relationships.

GLP compliance:

no

Key result

Parameter:

other: alerts

Value:

0

Remarks on result:

other: Skin sensitisation in mammals is non-sensitiser. No alert matched for QSAR predicted value. The substance is within the applicability domain of the model.

Interpretation of results:

other: Derek result: not sensitising

Conclusions:

Using Derek Nexus v6.0.1, the skin sensitising potential of the test item was estimated to be non-sensitiser. No alerts were matched. The substance is within the applicability domain of the model.

Executive summary:

The skin sensitising properties were estimated using Derek Nexus v6.0.1. The skin sensitising potential of the test item was estimated to be non-sensitiser based on the described QSAR method (Derek, 2018).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro studies covering three key steps of the adverse outcome pathway (AOP) for skin sensitisation as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. Direct Peptide Reactivity Assay (DPRA) according to OECD TG 442C, KeratinoSens according to OECD TG 442D and U-Sens according to OECD TG 442E were conducted.

DPRA

A study according to OECD TG 442C - Direct Peptide Reactivity Assay (DPRA) was conducted under GLP to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with SPCC or SPCL for 24 hours at 25 °C, the relative peptide concentration was determined by HPLC-PDA detection at 220 and 258 nm. As reference control cinnamic aldehyde was used. The test item was tested at test concentration of 78.2 mM and the reference item at 100 mM. As a vehicle acetonitrile (ACN) was used. Synthetic peptide control solutions of SPCC (containing Ac-RFAACAA-COOH) and SPCL (containing Ac-RFAAKAA-COOH) were prepared in ACN. Co-eluent samples containig only the test item and the respective buffer were also tested under the same conditions. After the incubation period samples were injected directly into the HPL-PDA system. The concentration of SPCC or SPCL was photometrically determined at 220 and 258 nm. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. Following this model, the mean % SPCC depletion of the test item was 0.46 % and the mean % SPCL depletion was 0 %. The average % depletion of the test item was determined to be 0.23 %. The reference control showed mean % SPCC depletion of 78.3 % and a mean % SPCL depletion of 52.8 %. All validity criteria were met and the test is considered as valid. As a precipitation was observed after adding the peptide solutions to the test item, the result is regarded as 'inconclusive for dendritic cells activation (due to precipitation)'.

KeratinoSens

The in vitro KeratinoSens™ assay according to OECD 442D enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In the present study the test substance was dissolved in DMSO to a final concentration of 200 mM.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 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%).Cells were incubated with the test item for 48 h at 37 °C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction compared to the vehicle control. Under the condition of this study the test item is, therefore, considered as non sensitiser. The controls confirmed the validity of the study. The calculated EC1.5 was between 5 and 125 µM (70 µM in experiment 1; 66 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (7.9 % in experiment 1; 8.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 2.33 and in the second experiment it was 2.55, thus, the criteria were fulfilled. 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 of this test up to the cytotoxicity limit.

U-Sens

This in vitro U937 cell line activation Test (U-Sens™) assay was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP). The test item was dissolved in DMSO at concentration of 50 mg/mL and administered to U937 cells for 45 ± 3 hours.In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL and 200, 120, 70, 20, 15, 10 and 7.5 µg/mL in experiment 1 and 2, respectively, in the 96-well plate (final concentration DMSO of 0.4 %).Positive and negative control ran parallel.FITC-conjugated antibodies were used for both IgG1 and CD86 staining. The test itemwas evaluated for the ability toincrease the expression levels of CD86 cell surface marker. No precipitation of the test item at the end of the experiments was observed. The test item showed no toxicity and the viability of the cells was higher than 70 % in all test concentrations, therefore, no CV70 values could be calculated andconsidered to be higher than 200 µg/mL. A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 for experiment 1 was 11 µg/mL and for experiment 2 was 15 µg/mL, respectively. All validity criteria were met and the test is considered as valid. Based on the obtained results from the two experiments, it can be considered that the test item has a skin sensitizing potential based on the key event "activation of dendritic cells". However, the extreme bell-shaped dose-response curve casts doubts on the result reliability. The cell activation response at higher concentrations reverted back to vehicle levels in the absence of any cytotoxicity.

In the DPRA assay, the test item showed no reaction with the peptides but precipitation was observed after adding the peptide solutions to the test item. As the test does not allow a clear conclusion if the test item is protein binding or not, the test is considered as equivocal. In the KeratinoSens assay, the test item was determined to be negative with regard to the activation of keratinocytes. In the U-Sens assay, the test item showed a sensitizing potential with regard to the activation of dendritic cells, however the dose-response was unusal and there was no relevant response at high concentrations in the absence of cytotoxicity.

QSAR Estimations

Based on the results of the in chemico/in vitro tests, a clear overall conclusion on the skin sensitizing potential of the test item could not be made. Therefore, two QSAR predictions were additionally performed. Two models were used to estimate the skin sensitizing potential of the test item. Using DEREK v6.0.1 the test item was estimated as non-sensitising substance. No alerts for sensitising were predicted. In the Danish Q(SAR) Database the sensitising potential of the test item was also predicted as negative.

Based on the results of the weight of evidence, it can be stated that the test item has no skin sensitising potential.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified as a skin sensitiser under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.