(carboxymethyl)dimethyl-3-[(1-oxotetradecyl)amino]propylammonium hydroxide

Endpoint summary

Description of key information

An in vitro skin sensitisation test battery is available for the test substance

key event 1: molecular interaction with skin protein: OECD 442C (RL1): negative

key event 2: inflammatory response in keratinocytes: OECD 442D (RL1): negative

key event 3: activation of dendritic cells: OECD442E (h-CLAT, RL1): positive

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 Jan - 11 Feb 1984

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

positive control missing, substance purity not given

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted in 1992

Deviations:

yes

Remarks:

no reliablity check was performed

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted prior to the current requirement in Regulation (EC) 1907/2006 to perform an LLNA study (OECD 429) as the preferred in vivo skin sensitisation study.

Species:

guinea pig

Strain:

not specified

Remarks:

albino

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Weight at study initiation: males: 237 - 315 g; females: 235 - 295 g
- Housing: individual in suspended stainless steel cages, fitted with wire mesh floors and fronts
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 40
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

propylene glycol

Concentration / amount:

20%

Day(s)/duration:

1

Adequacy of induction:

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

Route:

epicutaneous, occlusive

Vehicle:

other: vaseline

Concentration / amount:

10%

Day(s)/duration:

48 hours

Adequacy of induction:

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

Route:

epicutaneous, semiocclusive

Vehicle:

other: vaseline

Concentration / amount:

5% or 1%

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20 animals (10 male and 10 female) for test group and 10 animals (5 male and 5 female) for control group

Details on study design:

RANGE FINDING TESTS: no data available

MAIN STUDY
A. INDUCTION EXPOSURE
Induction is effected in a two-stage operation consisting of, firstly, three pairs of intradermal injections made simultaneously and, secondly, one week later, a closed patch exposure performed over the injection sites. For this purpose an area of c. 24 cm² of dorsal skin in the shoulder region is clipped free of hair with electric clippers.

Intradermal injections:
Three pairs of intradermal injections are made in the clipped area.
The preparations injected into the skin of the test animals are the following:
- sites a(left) and a(right): Freund’s Complete Adjuvant (FCA)
- sites b(left) and b(right): a dilution of the test substance in a suitable carrier (propylene glycol)
- sites c(left) and c(right): a dilution of the test substance in a mixture of FCA and carrier (1:1).
The preparations injected into the skin of the control animals are the following:
- sites a(left) and a(right): FCA
- sites b(left) and b(right): carrier
- sites c(left) and c(right): a mixture of FCA and carrier (1:1).

Skin readings are made 24 h after the treatment.

Topical application:
One week after induction by the intradermal injections the same area of the skin of the animals is closely shaved again. The test animals are treated as follows:
A 2 x 4 cm patch of Whatman No 3 MM filter paper is loaded with the appropriate mixture of the test substance and carrier. The patch is placed over the sites of the intradermal injections and covered with a piece of PVC foil and a piece of Leukopor hypo-allergic paper bandage. This, in turn, is secured with a 7.5 cm wide Tensoplast bandage. The dressing is left in place for 48 h. The control animals are similarly treated with patches with carrier (vaseline) only.
Skin readings are made after removal of the patches.


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after topical induction
- Exposure period: 24 hours
- Test groups: test substance
- Control group: test substance
- Site: left flank
- Concentrations: 5 or 1%
- Evaluation (hr after challenge): 24 and 48 hours

Positive control substance(s):

not specified

Positive control results:

no data available

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

5% at challenge

No. with + reactions:

1

Total no. in group:

9

Clinical observations:

1 male with score 1 for erythema and edema, respectively

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

5% at challenge

No. with + reactions:

0

Total no. in group:

9

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

20% intradermal induction, 10% topical induction, 5% at challenge

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

1 male with edema score of 1 and one male with erythema and edema score 1

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

20% intradermal induction, 10% topical induction, 5% at challenge

No. with + reactions:

0

Total no. in group:

20

Group:

positive control

Remarks on result:

not measured/tested

One control female died on Day 9 of the study because of a prolapse of the rectum. All other animals remained in good health during the experimental period.

The challenge treatment with a 1% dilution - a concentration which is one fifth of the non-irritant concentration - did not elicit any sensitisation reaction under the conditions of the test.

Interpretation of results:

study cannot be used for classification

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2015

Deviations:

yes

Remarks:

Demineralized water was used instead of HPLC grade or Millipore Milli-Q grade water. This was seen as uncritical, because the reference controls showed that the used water has no negative impact on the test system.

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Mainz, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

TEST SYSTEM
Peptides with >= 95% purity, synthesized be "RS synthesis" were used
- Peptide stock solution preparation: Stock solutions of cysteine and lysine peptides were freshly prepared.
Cysteine-containing peptide:
- Concentration: 0.667 mM
Lysine-containing peptide:
- Concentration: 0.667 mM

VEHICLE CONTROL
- Substance: Water
- Justification for selecting vehicle: The test substance was soluble in the vehicle at the test concentration of 100 mmol/L.

POSITIVE CONTROL
- Substance: Cinnamaldehyde (CAS 104-55-2) for the Cys-peptide and 2,3-Butanedione (CAS 431-03-8) for the Lys-peptide
- Preparation: The positive controls were prepared as a 100 mM solution in acetonitrile.

CO-ELUTION CONTROL:
- Sample prepared from the respective peptide buffer and the test item, but without peptide.

REFERENCE CONTROLS:
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, in total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples, and was used to calculate the peptide depletion. Additionally, a solvent control containing water instead of test item solution was prepared in triplicate (set C(water)) and also incubated and analysed together with the samples. Set C (water) was used for calculation of the depletion of the test item. For the reference Control C (water) in the Cys peptide assay 750 µL Cys-peptide stock solution, 200 µL acetonitrile and 50 µL HPLC grade water was used. For the reference control C (water) in the Lys-peptide assay 750 µL Lys-peptide stock solution and 250 µL HPLC grade water were mixed.

TEST SUBSTANCE PREPARATION: The test substance was prepared as a 100 mM solution in water.

INCUBATION CONDITIONS:
- Peptide ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25°C
- Duration of treatment / exposure: 22 hours with Cys-peptide and 22 hours and 20 minutes for Lys-peptide

NUMBER OF REPLICATES: 3

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:
- Wavelength: 220 nm for quantitation and 258 nm as indicator for co-elution

Positive control results:

The positive control revealed a depletion of cysteine and lysine peptides (81.7% and 23.91, respectively). According to the cysteine 1:10/lysine 1:50 prediction model “high reactivity” was derived for the positive control, leading to a DPRA prediction of “positive“.

Key result

Run / experiment:

other: Cystein/Lysine

Parameter:

other: mean peptide depletion (%)

Value:

0.54

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: DPRA prediction: negative

Run / experiment:

other: Cystein

Parameter:

other: mean peptide depletion (%)

Value:

0.65

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: Lysine

Parameter:

other: mean peptide depletion (%)

Value:

0.42

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
a) The mean peptide concentration of all solvent controls (Reference A and Reference C (ACN and water)) were with 0.51 mM, 0.50 mM and 0.48 mM for the Cys-peptide and 0.50 mM, 0.50 mM and 0.48 mM for the Lys-peptide in the acceptable range of 0.50 ± 0.05 mM.
b) The variation coefficients (RSD) of the measured values of Reference controls B and C in acetonitrile were in the acceptable range with 1.4% for the Cys-peptide and 0.5% for the Lys-peptide, respectively. RSD of reference controls C in water are not evaluated.
- Acceptance criteria met for positive control: c) The mean peptide depletion with 81.70% and a standard deviation of 0.41% of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0% and < 14.9%, respectively, for the Cys-peptide.
b) The mean peptide depletion with 23.91% and a standard deviation of 3.92% of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0% and < 11.6%, respectively, for the Lys-peptide.
- Acceptance criteria met for variability between replicate measurements: The standard deviation for the test item replicates with 0.79% was < 14.9% for the percent cysteine depletion for the test item. The standard deviation for the test item replicates with 0.72% was < 11.6% for the percent lysine depletion for the test item.
- Range of historical values: The results of this study were within the range of historical control values.

Interpretation of results:

other: no skin sensitising potential based on the key event “protein reactivity”

Conclusions:

There is regulatory acceptance in the EU for the application of the Direct peptide reactivity assay to address key event 1: peptide/protein binding in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test substance did not show reactivity towards selected proteins. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E: In vitro skin sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

adopted in 2017

Deviations:

yes

Remarks:

The reactivity check for the THP-1 cells wasn´t performed 2 weeks, but 4 weeks after thawing. The cells didn’t have a higher passage than 30 and were no longer in culture than 2 months. This is uncritical and doesn’t have any influence on the results.

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Mainz, Germany

Type of study:

activation of dendritic cells

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Controls:
- Solvent control: RPMI 1640: 1% in culture medium
- Positive control: 2,4-dinitrochlorobenzene (DNCB, CAS 97-00-7): 4 µg/mL

Pre-Test: The solubility of the test item was determined in a non-GLP pre-test in RPMI 1640. The test item is soluble in medium at the concentrations 100 mg/mL and 200 mg/mL. Therefore, RPMI 1640 was used as solvent in the test. A possible autofluorescence of the test item was determined using a 2475 Multi-λ Fluorescence Detector and an excitation wavelength of 488 ± 5 nm. No emission was detected between 500 - 700 nm. Therefore, it is assumed that the test item has no influence on the result of the assay due to autofluorescence.
Dose selection: A preliminary cytotoxicity test was performed to determine the concentrations to be used for the CD86/CD54 expression measurement in the main experiments. The following 8 concentrations of the test item were tested with an incubation time of 24 h in the pre-test: 1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.3 µg/mL, 15.6 µg/mL and 7.8 µg/mL.

Main experiments:
Three independent experiments were performed in the same way (experiment I, II, III). Experiment I was invalid and not used for evaluation.
Cell line: THP-1 Cell Line from CLS (Eppelheim, Germany); cells of passage 20 and 21 were used.

Reactivity check: Four weeks after thawing, a reactivity check of the cells was performed. For that, the two positive controls 2,4-dinitrochlorobenzene (DNCB) (CAS 97-00-7, ≥ 99% purity, test concentration: 4 µg/mL) and nickel sulfate (NiSO4) (CAS 10101-97-0, ≥ 99% purity, test concentration: 200 µg/mL) as well as the negative control, lactic acid (LA) (CAS 50-21-5, ≥ 85% purity, test concentration: 1000 µg/mL) were used. The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore, the cells were found to be suitable for the experiments. For the pre-test as well as the experiments only cells which have successfully passed the reactivity check were used.

Dose selection: In the first pre-test none of the controls induced a cytotoxic effect, the cell viabilities of medium and solvents controls were higher than 90%, and the pretest was valid. Since all tested concentrations of the test item were cytotoxic (≤ 50%), the pre-test had to be repeated using lower concentrations: 0.06 µg/mL, 0.12 µg/mL, 0.24 µg/mL, 0.49 µg/mL, 0.98 µg/mL, 1.95 µg/mL, 3.90 µg/mL, 7.80 µg/mL. In the second pre-test none of the controls induced a cytotoxic effect, the cell viabilities of medium and solvents controls were higher than 90%, and the pre-test was valid.
The logCV75 was calculated and is: 0.68.
This value corresponds to a CV75 of 4.84 µg/mL. On the basis of the CV75 the maximum test substance concentration for the experiments (1.2 * CV75) was calculated and is: 5.80 µg/mL. Eight test item concentrations were tested in each experiment (5.8 µg/mL, 4.8 µg/mL, 4.0 µg/mL, 3.4 µg/mL, 2.8 µg/mL, 2.3 µg/mL, 1.9 µg/mL, 1.6 µg/mL).

Procedure: 1000000 cells / well of a 24-well plate were exposed to each concentration of the test substance for 24 h at 37.0 ± 1.0 °C and 5.0 ± 0.5% CO2. During treatment the plates were closed with a sealing tape to avoid cross-contamination. Following treatment, the cells were transferred into sample tubes, collected by centrifugation (250 * g for 5 min) and then washed twice with 1 mL staining buffer. After that, the cells were resuspended in 600 µL blocking solution and incubated on ice for 15 min. Then, 180 µL of the cell suspension was added in three wells of a 96-well round bottom plate respectively. The plates were centrifuged at 250 * g for 5 min and the supernatant was discarded. After that, 50 µL of diluted FITC-labelled antibodies (anti-CD86, anti-CD54 or mouse IgG1) was added in one of the three wells respectively and the plate was incubated on ice for 30 min in the dark. Before use, the antibodies were diluted 3:25 v/v for CD86, 3:50 v/v for CD54 with staining buffer. After the incubation on ice, the cells were washed 3 times with 150 µL staining buffer (centrifugation: 250 * g for 5 min) before 150 µL staining buffer as well as 7.5 µL PI working solution was added to each well. Afterwards the plate was incubated for 15 min on ice and 5 min at room temperature in the dark before measurement at the flow cytometer (BD FACS LyricTM). FITC-Detection: (Excitation: 488 nm / Detection filter 527/32; corresponding to the FL-1 channel). In total 10,000 viable cells (PI negative) were acquired and analysed.

Replicates: 3

Positive control results:

DNCB showed and RFI for CD86 of 458% and 728% in experiment II and III, respectively. The cell viability was at 84.7% and 88.4%, respectively. The RFI for CD54 was 380% and 548% in experiments II and II, respectively. The cell viability was at 84.7% and 87.7%, respectively. Thus, both meet the acceptance criteria.

Key result

Run / experiment:

other: CD86, experiment II

Parameter:

other: RFI (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 5.8 µg/mL and cell viability of 65%

Key result

Run / experiment:

other: CD54, experiment II

Parameter:

other: RFI (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 5.8 µg/mL and cell viability of 63%

Key result

Run / experiment:

other: CD86, experiment III

Parameter:

other: RFI (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 5.8 µg/mL and cell viability of 73%

Key result

Run / experiment:

other: CD54, experiment III

Parameter:

other: RFI (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values: The results of this study were within the range of the historical values.

Interpretation of results:

other: skin sensitising potential based on the key event “activation of dendritic cells”

Conclusions:

There is regulatory acceptance in the EU for the application of the h-CLAT test method to address key event 3: monocytic/ dendritic cell response in the skin sensitisation Adverse Outcome Pathway.
Under the conditions of the test, the test substance induces dendritic cell activation. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Mainz, Germany

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Controls:
- Negative control: DL-Lactic Acid: 5000 µM
- Positive control: p-Phenylenediamine: 60 µM (In difference to the OECD 442D guideline, Ethylene glycol dimethylacrylate (EGDMA) was not used as positive control, but p-Phenylenediamine was. This is uncritical since the guideline indicates that other suitable positive controls, preferentially providing EC 1.5 values in the mid-range, may be used if historical data are available to derive comparable run acceptance criteria.
- Solvent control: DMEM

Cytotoxicity Range Finder Test: A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration range applicable for the main experiments. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. This yellow tetrazole is reduced to purple formazan in viable cells and can therefore be used for assessing the cell metabolic activity and therefore the cell viability. A reduction of the viability below 70% is defined as a cytotoxic effect. In the CRFT the following 12 nominal concentrations of the test item were tested: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1000 µM, 2000 µM. The exposure time was 48 h.

Dose selection: In accordance with the OECD guideline 442D, the maximum final test item concentration should be 2000 µM. For a test chemical which has no defined molecular weight, the final test item concentration 2000 µg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility). Since a cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations were chosen for experiment I and II: 1.21 µM, 1.45 µM, 1.74 µM, 2.09 µM, 2.51 µM, 3.01 µM, 3.62 µM, 4.34 µM, 5.21 µM, 6.25 µM, 7.50 µM, 9.00 µM. In the main experiments, a reduction of the viability below 70% is considered as cytotoxic and the concentration that is cytotoxic may not be evaluated for luciferase induction.

Main experiments:
Two independent experiments were performed in the same way (experiment I and II).
Cell line: LuSens cell line was obtained from the BASF SE (Ludwigshafen, Germany).
Procedure: About 10000 cells were seeded in two 96 well plates (one for viability and one for luciferase induction measurment) and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 in a humidified atmosphere for 24 h in both experiments. After incubation time medium was replaced with test substance or control substance. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross-contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1°C in a humidified atmosphere containing 5.0 ± 0.5% CO2. For the evaluation of the viability, MTT solution was added to each well after removal of all test substance or control solutions. The plate was incubated for 2 h at 37 ± 1°C and 5.0 ± 0.5% CO2 in a humidified atmosphere. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow colour) to its insoluble formazan (purple colour) in living cells and therefore indicates the amount of living cells.
For the evaluation of the Luciferase expression, after the 48 h exposure period, all solutions were removed from the wells and the cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Afterwards 100 µL per well of a Lysis buffer was added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 µL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 µL per well was transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer.

Replicates: 3

Calculation of Relative Viability
The calculation of the relative Viability [%] was performed as follows:
All wells were corrected = OD570 value - OD690 value
For the following calculation only the corrected values were used:
Viability = [( Vsample - Vblank) / (Vsolvent - Vblank)] * 100
Vsample= MTT-absorbance reading in the test chemical well
Vblank = MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent = is the average MTT-absorbance reading in wells containing cells and solvent
Afterwards the mean value of the single replicates was calculated.

Calculation of CV75
The CV75-value (relative survival rate) was calculated by linear interpolation. This value is the substance concentration at which cell viability is 75% compared to the control.
The CV75 was calculated as follows:
CV75 = (Cb - Ca) * [(75 - Vb) / (Vb - Va)] + Cb
Ca = lowest concentration in µM or µg/mL with > 75 % cell viability
Cb = highest concentration in µM or µg/mL with < 75 % reduction in viability
Va = % viability at the lowest concentration with > 75 % cell viability
Vb = % viability at the highest concentration with < 75 % cell viability

Calculation of Luciferase fold induction
Fold induction = [(Lsample - Lblank) / (Lsolvent - Lblank)]
Lsample = luminescence reading (RLU) in the test chemical well
Lblank = luminescence reading in blank well containing no cells and no treatment
Lsolvent = average luminescence reading in wells containing cells and solvent
Afterwards the mean value of the single replicates was calculated.

Positive control results:

Experiment I: 4.7-fold induction and 71.5% relative viability; Experiment II: 4.6-fold induction and 78.0% relative viability

Key result

Run / experiment:

other: experiment I

Parameter:

other: luciferase induction

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentrations of 5.21µM, 6.25µM, 7.5µM, 9.0 µM were not used for the final evaluation due to cytotoxicity

Key result

Run / experiment:

other: experiment II

Parameter:

other: luciferase induction

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentrations of 6.25µM, 7.5µM, 9.0 µM were not used for the final evaluation due to cytotoxicity

Other effects / acceptance of results:

CRFT:
No cytotoxic effect was observed at the controls as well as the test item concentrations 0.98 µM to 3.91 µM. The viability values were all ≥ 72%. In all other test item concentrations (7.81 µM to 2000 µM) cytotoxic effects were observed with relative viability values below 59%. No precipitation was observed.
The CRFT was valid and could be used for the determination of the concentrations to be tested in the main experiments. The CV75 value is 3.52 µM.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes: Negative control: Experiment I: 0.9-fold induction and 107.7% relative viability; Experiment II: 0.9-fold induction and 100.5% relative viability; Growth control: Experiment I: 1.0-fold induction and 99.6% relative viability; Experiment II: 1.0-fold induction and 99.7% relative viability
- Acceptance criteria met for positive control: Yes: Experiment I: 4.7-fold induction and 71.5% relative viability; Experiment II: 4.6-fold induction and 78.0% relative viability
- Acceptance criteria met for variability between replicate measurements: Yes: The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells was 11.13% for experiment I and 6.40% for experiment II. 8 and 9 concentrations in experiment I and experiment II, respectively, were within viability limits. 4 and 3 concentrations in experiment I and II, respectively, were cytotoxic.

The luciferase induction was not above or equal to 1.5 -fold in comparison to the solvent control in more than 2 consecutive non-cytotoxic test item concentrations in experiment I
and II. Therefore, the test substance is considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential) under the conditions of the LuSens test.

Interpretation of results:

other: no skin sensitising potential based on the key event “inflammatory response in keratinocytes”

Conclusions:

There is regulatory acceptance in the EU for the application of the ARE-Nrf2 luciferase test method to address key event 2: keratinocyte response in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test substance did not have a keratinocyte activating potential. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the evaluation of the skin sensitising effect of the test substance three in vitro/in chemico skin sensitisation tests were performed addressing the three key events according to Reach Regulation (EC) No 1907/2006 Annex VII.

In chemico: Direct Peptide Reactivity Assay

The skin sensitising potential of the test substance was assessed in a Direct Peptide Reactivity Assay (DPRA) performed according to OECD guideline 442C and in compliance with GLP (LAUS, 2019).The study was performed in order to evaluate the reactivity of the test substance towards cysteine (Cys-) and lysine (Lys-) containing peptides. The test substance was incubated for 22 h at 25 °C together with Cys-peptide and 22 h 20 min with the Lys-peptide, respectively. The peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test substance with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test substance were incubated and measured simultaneously.
One experiment was performed and it was valid for the Cys- and Lys-peptide assay. The
results were evaluated according to the Cysteine 1:10/Lysine 1:50 prediction model. The peptide depletion values are as following: Cys-peptide: 0.65%, Lys-peptide: 0.42%. This results in a mean peptide depletion of 0.54%.

In conclusion, the DPRA prediction is “negative” with no or minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test substance possesses no or minimal skin sensitisation potential.

 

In vitro: Activation of keratinocytes

The activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test in the transgenic LuSens cell line performed according to OECD guideline 442D and in compliance with GLP (LAUS, 2019).The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test substance. Based on the results of this test the concentrations for the experiments were determined. In the experiments, the highest nominal applied concentration (9.00 µM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions was prepared. Precipitation of the test substance was not visible in any of the experiments. DMEM (final concentration: 1%) was used as solvent control and medium no. 3 as growth
control. Lactic acid (5000 µM) was used as negative control and p-Phenylenediamine (60 µM) as positive control. No substantial and reproducible dose-dependent increase in luciferase induction equal or above 1.5 fold was observed in both experiments up to the maximal tested non-cytotoxic concentration of the test substance. Therefore, under the experimental conditions of this study, the test substance was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).

In vitro: Activation of dendritic cells

The capability of dendritic activation of the test substance was investigated in a human cell line activation test (h-CLAT) performed according to OECD guideline 442E and in compliance with GLP (LAUS, 2019).This in vitro study was performed to assess the sensitising potential of the test substance by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. In total two pre-tests and three experiments (experiment I - III) with a treatment period of 24 hours were performed whereby experiment I was invalid and had to be repeated. Therefore, in total two valid experiments (experiment II and III) were performed. For the experiments, the highest nominal applied concentration (5.8 µg/mL) was chosen based on the results obtained in the pre-tests. A geometric series (factor 1.2) of 7 dilutions was prepared. As solvent control for the test item, RPMI 1640 was used in a final concentration of 1% in culture medium. As positive control, 2,4-dinitrochlorobenzene was used.
The evaluated experimental points and the results are summarised in the following:
Experiment II: The RFI of CD86 was ≥ 150% and the RFI of CD54 was ≥ 200% in the highest test item concentration (5.8 µg/mL).
EC200 (CD54): 5.18 µg/mL; EC150 (CD86): 5.02 µg/mL.
Experiment III: The RFI of CD86 was ≥ 150% and the RFI of CD54 was ≤ 200% in the highest test item concentration (5.8 µg/mL).
EC150 (CD86): 5.29 µg/mL.
Since both runs are positive for CD86 in the highest tested concentration, the test item is considered as “positive”. Therefore, under the experimental conditions of this study, the test substance was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and therefore to be a potential skin sensitizer.

In conclusion, testing of the test substance was done for the three key events of the adverse outcome pathway (AOP) for skin sensitization. Based on the “2 OUT OF 3” weight of evidence / integrated testing strategy approach to skin hazard identification (ENV/JM/MONO(2016)29/ANN1) the following conclusion can be drawn:

Testing for the first and second key event (namely protein reactivity and activation of keratinocytes, respecitively) revealed clear negative results. Testing for the third molecular key event (namely activation of dendritic cells) revealed activation of CD86 but no activation of CD54. Leading in sum to a positive result. Following the WoE approach with one positive and two negative valid tests, the test substance is considered to be no skin sensitizer and, accordingly, does not meet CLP/GHS criteria and no classification is required according to Regulation (EC) No. 1272/2008.

Supporting this conclusion is an in vivo test in guinea pig maximisation test (CIVO-TNO, 1984) where no sensitising potential was observed. However, this study was considered to be invalid because no positive control was included or reported.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.