Cyclooctane

Endpoint summary

Description of key information

Precipitations were observed upon adding test item to buffer solution. Therefore, the negative result observed cannot be used in an assessment of skin sensitisation potential, as mentioned in the OECD Test Guideline No. 442C, and the DPRA results are considered inconclusive (reference 7.4.1-1).

The test item did not activate the LuSens cells up to a concentration of 187 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP (reference 7.4.1-2).

The test item activated U-937 cells and is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP) (reference 7.4.1-3).

The test item was found to be a skin sensitiser in a LLNA and an EC3 value of 57.1 % (w/w) was derived (reference 7.4.1-4).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-07-13 to 2021-09-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase LuSens test method

Details of test system:

Lusens transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution and serial dilutions
On the day of the experiment (immediately before treatment) the test item was dissolved in DMSO to prepare a stock solution. The highest test concentration for the first dose finding assay was 2000 μM and 300 μM in the second dose finding assay. For the MTT test (dose finding assay) twelve concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from the highest concentration.
- Preparation of the positive controls
Positive control: EGDMA (final concentration 120 μM), solved in treatment medium including 1 % (v/v) DMSO
- Preparation of the solvent, vehicle and negative controls
Medium control: Treatment medium
Solvent control for the positive control: DMSO, final concentration 1 % (v/v) in treatment medium
Negative control: Lactic acid (final concentration 5000 μM), solved in treatment medium including 1 % (v/v) DMSO

DOSE RANGE FINDING ASSAY:
- Highest concentration used: The highest test concentration for the first dose finding assay was 2000 μM and 300 μM in the second dose finding assay.
- Solubility in solvents: Test item was sufficiently soluble.
- Cytotoxicity assessment performed: yes, calculation of CV75 was done
- Final concentration range selected on basis of:
With reference to the CV75 parameter of the first dose finding assay the highest tested concentration in main experiment 1 was 187 μM. The highest tested concentration for the main experiment 4 and 5 was 183 μM without reference to the CV75 values of the dose finding assays.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: test item: 3 per concentration; solvent control: 24; positive control: 5; negative control: 6; treatment medium control: 12
- Number of repetitions: three main experiments (with valid results); In total 5 main experiments were conducted.
- Test chemical concentrations:
first main experiment: 75.2, 90.2, 108, 130, 156, 187 μM
main experiment 4 and 5: 35.5,42.6, 51.1, 61.3, 73.5, 88.3, 106, 127, 153, 183 μM

- Application procedure: 24 h ± 30 min after seeding of the cells, seeding medium were removed and 150 μL of treatment medium were distributed in each well. Thereafter, 50 μL of the test item and control dilutions and the medium control were added into the corresponding wells.
- Exposure time: 48 ± 1 h
- Study evaluation and decision criteria used:
The luciferase fold induction is calculated using the following formula (OECD 442D): Fold induction=(Lsample-Lblank)/(Lsolvent-Lblank)
where
Lsample is the luminescence reading in the sample well (samples: test item concentrations, medium, negative and positive control)
Lblank is the luminescence reading in the blank well without cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent control
The arithmetic mean of the luciferase fold induction was calculated for each sample.
- Description on study acceptance criteria:
The following acceptance criteria should be met in the LuSens test method (OECD 442D):
• The average luciferase activity induction obtained with the positive control, 120 μM EGDMA should be ≥2.5, and the positive control should have a relative cell viability ≥70 % as compared to the solvent control.
• The average luciferase activity induction obtained with the negative control, 5000 μM lactic acid, as well as the basal expression of untreated cells should be <1.5-fold as compared to the average solvent control.
• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20 % in each main experiment.
• At least three test item concentrations should have cell viability of at least 70 % relative to the solvent controls. Moreover, in case a result is to be considered negative, at least one concentration should be cytotoxic, i.e. have a cell viability <70 %, or the maximum concentration of 2000 μM (or 2000 μg/ mL for substances with no defined MW) should have been tested.
If a test item is tested with a concentration < 2000 μM (or 2000 μg/ mL for substances with no defined MW) and no luciferase induction as well as no cytotoxicity are observed, a second main experiment should be performed using e.g. a 1.44 serial dilution factor based on the CV75 (i.e. starting with 1.44 x CV75) instead of the 1.2 serial dilution factor. If in the second main experiment cytotoxicity and luciferase induction are still not observed, a third main experiment should be conducted with the maximum concentration of 2000 μM (or 2000 μg/mL for substances with undefined MW). This main experiment should then be confirmed by performing a fourth main experiment (OECD 442D).

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): The passage numbers of the used LuSens cells were 5 and 9 in the cytotoxicity tests and 7, 11 and 13 in the LuSens test for the main experiments 1, 4 and 5, respectively. Each treatment well of a 96 well microtiter plate was seeded with 100 μL cell suspension (9000 to 11000 LuSens cells per well), except the well of the blank control.
- Incubation conditions: 37 ± 1.5 °C and 5.0 ± 0.5 % CO2
- Washing conditions: At the end of the incubation period (48 ± 1 h), the treatment medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+.
- Precipitation noted: no

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: The absorption and luminescence measurement of the LuSens samples were conducted with a Multimode Reader (TriStar2 LB 942) by Berthold Technologies GmbH Co KG, Germany. The experimental values were determined and calculated using the software MikroWin 2010, Version 5.19. Control tests were conducted as part of the experiments.
- Plate used: 96 well microtiter plate
- Measurement of the luciferase activity: The Steady-Glo® mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part of Steady-Glo® mix.
At the end of the incubation period (48 ± 1 h), the treatment medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 sec per well.

DATA EVALUATION
- Cytotoxicity assessment:
The quantity of formazan is presumably directly proportional to the number of viable cells, as monitored by the absorbance. The relative absorbance (= cell viability) as compared to the solvent control is calculated using this formula: relative absorbance [%] = 100 × ((absorbance sample – absorbance blank)/(absorbance solvent control – absorbance blank)).
absorbance sample is the MTT-absorbance reading in the sample well
absorbance blank is the MTT-absorbance reading in the blank well (no cells and treatment)
absorbance solvent control is the average MTT-absorbance reading in the wells with cells and solvent control
The arithmetic mean was calculated for each sample.
The CV75 value, a concentration showing 75 % of LuSens cell survival (25 % cytotoxicity), is calculated by using the following equation: CV75 = Conc.>75 – ((Conc. < 75) x (%>75 – 75)/(%>75 - %<75))
Conc. >75 = max. measured concentration with the % of solvent control >75 % ≡ a)
Conc. <75 = min. measured concentration with the % of solvent control <75 % ≡ b)
% >75 = relative absorbance at a) in %
% <75 = relative absorbance at b) in %
The values stated in the report are rounded values.
- Prediction model used:
If the luciferase induction is ≥1.5-fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥70%) and at least 3 tested concentrations are non-cytotoxic, the main experiment of the LuSens prediction is considered positive.
If these conditions are met in 2 of 2 or in 2 of 3 main experiments, the LuSens prediction is considered positive, otherwise the LuSens prediction is considered negative.
A negative result obtained with a test item that does not form a stable dispersion and was not tested up to 2000 μM (or 2000 μg/mL for test items with no defined MW) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive (OECD 442D).
If no clear dose-response curve or a biphasic dose-response curve is observed, the experiment should be repeated to verify whether this is specific to the test item or due to an experimental artefact. If the biphasic response (i.e. when the threshold of 1.5 is crossed twice) is reproducible in an independent verification experiment, the lower concentration of a ≥ 1.5 induction should be reported (i.e. when the threshold of 1.5 is crossed the first time).
Mono constituent test items with a Log Pow > 7 may be insoluble in the culture medium. However, if the test item is soluble or can be stably dispersed/suspended, the LuSens test may be performed.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

EGDMA (120 M) [442D]

Positive control results:

The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 7.08; ME 4: 4.14; ME 5: 3.57). The positive control had a relative cell viability ≥70 % as compared to the solvent control (ME 1: 99.43 %; ME 4: 109.16 %; ME 5: 113.23 %).

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: luciferase induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The luciferase induction was not above or equal to 1.5-fold compared to the solvent control in all tested concentrations.

Group:

test chemical

Run / experiment:

other: second cytotoxicity test

Parameter:

CV75 [442D and 442E]

Value:

47.7 µM

Group:

test chemical

Run / experiment:

other: first cytotoxicity test

Parameter:

CV75 [442D and 442E]

Value:

156.1 µM

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the LuSens with the OECD 442D guideline recommended proficiency substances was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 1.08; ME 4: 1.07; ME 5: 0.30 %), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 0.99; ME 4: 0.66; ME 5: 0.27).
- Acceptance criteria met for positive control: Yes. For details refer to “Positive control results”.
- Acceptance criteria met for variability between replicate measurements: Yes. The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 6.6 %; ME 4: 14.5 %; ME 5:10.4 %). At least three test item concentrations had a cell viability of at least 70 % relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability <70 %.

Please refer to attached document under "Overall remarks, attachments". 

Interpretation of results:

other: The test item is negative for the second key event of the skin sensitisation AOP.

Conclusions:

The test item did not activate the LuSens cells up to a concentration of 187 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.

Executive summary:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens; OECD TG 442D) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of the skin sensitization Adverse Outcome Pathway (AOP)) of the test item.

To determine the dose range of test item concentrations, two dose finding assays were performed. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 μM (threshold of cytotoxicity: <70 % cell viability) in the first cytotoxicity assay and at a concentration of 178 μM in the second cytotoxicity assay. The CV75 value of the first cytotoxicity test was calculated as 156.1 μM and 47.7 μM in the second one.

The test item was tested in 5 independent main experiments, of which 2 main experiments yielded in invalid results, since acceptance criteria regarding cytotoxicity was not met. Only the results of valid main experiments (main experiment 1, 4 and 5) are reported and the original numbering is used.

The following concentrations of the test item were tested in the first main experiment: 75.2, 90.2, 108, 130, 156, 187 μM.

After treatment with the test item for 48 ± 1 h in the main experiment 1 the luciferase induction is <1.5-fold compared to the solvent control in 5 tested concentrations. Since the test item is considered negative, the acceptance criteria regarding testing at least one cytotoxic concentration is met.

To characterize the test item concentrations in more detail, two additional main experiments (main experiment 4 and 5) were conducted with 10 test item concentrations. The following concentrations of the test item were tested: 35.5,42.6, 51.1, 61.3, 73.5, 88.3, 106, 127, 153, 183 μM.

After treatment with the test item for 48 ± 1 h the luciferase induction was not above or equal to 1.5-fold compared to the solvent control in all tested concentrations. Since the test item is considered negative, the acceptance criteria regarding testing at least one cytotoxic concentration is met.

The LuSens prediction for the test item is considered negative.

The acceptance criteria were met:

- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 7.08; ME 4: 4.14; ME 5: 3.57).

- The positive control had a relative cell viability ≥70 % as compared to the solvent control (ME 1: 99.43 %; ME 4: 109.16 %; ME 5: 113.23 %).

- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 1.08; ME 4: 1.07; ME 5: 0.30 %), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 0.99; ME 4: 0.66; ME 5: 0.27).

- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 6.6 %; ME 4: 14.5 %; ME 5:10.4 %).

- At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability <70 %.

In conclusion, the test item did not activate the LuSens cells up to a concentration of 187 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-07-19 to 2021-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

U937 cell line activation test (U-SENS™)

Details of test system:

U-937 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical solution: On the day of the experiment, the test item was dissolved in DMSO to prepare a stock solution. Subsequently, dilutions in DMSO were prepared from the stock solution and further dilutions were prepared in culture medium.
- Preparation of the positive controls: Picryl sulfonic acid (TNBS), (CAS No. 2508-19-2, 1 M in water) final concentration 50 µg/mL, using culture medium as solvent
- Preparation of the solvent, vehicle and negative controls:
Solvent control: DMSO (CAS No. 67-68-5, purity ≥ 99 %) final concentration 0.4 % DMSO in culture medium
Negative control: Lactic acid (CAS No. 50-21-5, purity > 85 %), final concentration 200 µg/mL, using culture medium as solvent

DOSE RANGE FINDING ASSAY:
No dose range finder was conducted. Two test runs were performed and the doses for the second run were adjusted based on the first run.
- Highest concentration used: The highest test item concentration was 200 µg/mL in accordance with OECD Guideline 442E.
- Solubility in solvents: Test item was sufficiently soluble.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: test item: 2 per concentration; controls: 6
- Number of repetitions: 2
- Test chemical concentrations: first experiment: 1, 10, 20, 50, 100 and 200 µg/mL; second experiment: 1, 12.5, 15, 20, 25, 50 µg/mL
- Application procedure: Each volume (100 µL) of the dilutions of the test item, culture medium, positive, negative and solvent control was added to the cells according to the plate template. The dilutions of the test item were tested in two replicates (one labelled with anti-CD86 and one with mouse IgG1). The control groups were tested in six replicates (three labelled with anti-CD86 and three with mouse IgG1).
- Exposure time: 45 ± 3 hours
- Study evaluation and decision criteria used:
For each viable condition (“% viability mean” ≥ 70 %), the % of IgG1 positive cells was subtracted from the % of CD86 positive cells. The results were expressed as stimulation index (S.I.) and calculated as follows: 𝑆.𝐼. = ((% of CD86 treated cells − % of IgG1 treated cells)/ (% of CD86 control cells − % of IgG1 control cells)) × 100. The percentage of control cells (solvent/vehicle, i.e. complete medium or DMSO) was the mean of the 3 values obtained, unless one (outlier) was clearly out of the range of the other two. Cell viability = ((Number of living cell)/ (Total number of acquired cells))× 100. If possible, the CV70 value and the EC150 value are calculated in the U-SENS™ test method by log-linear interpolation using the following equitation: CV70 = C1+[(V1-70)/(V1-V2)×(C2-C1)]. Where: V1: is the minimum value of cell viability over 70 %; V2: is the maximum value of cell viability below 70 %; C1 and C2 are the concentrations showing the value of cell viability V1 and V2 respectively. EC150 = C1+[(150-S.I.1)/(S.I.2-S.I.1)×(C2-C1)]. Where: C1 is the highest concentration in µg/mL with a CD86 S.I. < 150 % (S.I.1); C2 is the lowest concentration in µg/mL with a CD86 S.I. ≥ 150 % (S.I.2)
- Description on study acceptance criteria:
The following acceptance criteria should be met when using the U-SENS™ method:
•At the end of the exposure period, the mean viability of the triplicate untreated U937 cells should be more than 90 % and no drift in CD86 expression is observed. The CD86 basal expression of untreated U937 cells had to be within the range of ≥2 % and ≤25%.
• When DMSO is used as a solvent, the validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells had to be >90 %. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. is smaller than 250 % of the mean of the triplicate CD86 S.I. of untreated U937 cells.
• The runs are considered valid if at least two out of three IgG1 values of untreated U937 cells fall within the range of ≥0.6 % and <1.5 %.
• The negative control is considered valid if at least two out of the three replicates are negative (CD86 S.I. <150 %) and non-cytotoxic (cell viability ≥70 %).
• The positive control is considered valid if at least two out of the three replicates are positive (CD86 S.I. ≥150 %) and non-cytotoxic (cell viability ≥70 %).

SEEDING AND INCUBATION
- Seeding conditions: On the day of the experiment (U-SENS™) directly before the treatment of the cells, a volume of 100 µL with a cell density of 0.49-0.51 × 10^5 U937 cells/well was seeded in each corresponding well of a 96-well flat bottom plate. Cells used were passaged 21 times or less.
- Incubation conditions: The treated U937 cells were incubated for 45 ± 3 hours at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Prior to incubation, plates are sealed with semi-permeable membrane, to avoid evaporation of volatile test items and cross-contamination between cells treated with the test item.
- Washing conditions: washed with approx. 100 µL of staining buffer (PBS with 5 % (w/v) FBS).

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: FACSCalibur, Becton Dickinson GmbH; The expression of cell surface antigens was analysed by flow cytometry using the software Cellquest Pro 6.0.
- Plate used: v-shape 96-well plate, not further specified
- Preparation for CD54 and/or CD86 expression measurements/cell staining: All cells were transferred according to the plate template in a v-shape 96-well plate, collected by centrifugation (approx. 200 × g, 5 min) and then washed with approx. 100 µL of staining buffer (PBS with 5 % (w/v) FBS). Thereafter, the cells were centrifuged, and the cell pellets were re-suspended in 100 µL staining buffer. The cells were stained with FITC-labelled anti-CD86 and mouse IgG1 (isotype control) and incubated light protected for 30 ± 5 min on ice. After staining with the antibodies, the cells were washed twice with 100 µL of staining buffer and once with 100 µL ice cold PBS. The cells were re-suspended in 100 µL ice cold PBS and transferred into microtubes according to the plate template. A volume of 300 µL PBS/tube was added. At least 10 min before the flow cytometry acquisition, 5 µL of a 7-AAD solution/tube was added. For measurement each microtube was placed in a proper cytometer tube and vortexed.

DATA EVALUATION
- Cytotoxicity assessment
- Prediction model used: According to OECD 442E (2018). The test item was tested in at least four concentrations and in at least two independent runs to derive as single prediction (CD86 NEGATIVE or CD86 POSITIVE). The individual conclusion of a U-SENS™ is considered NEGATIV (N) if the S.I. of CD86 is less than 150 % at all non-cytotoxic concentrations (cell viability ≥ 70 %) and if no interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 % ) is observed.
In all other cases: If S.I. of CD86 ≥ 150 % or interference is observed, the individual conclusion of a U-SENS™ run is considered POSITIVE (P).
• A U-SENS™ prediction will be considered negative if the first two independent runs are negative, a third run is not necessary.
• A U-SENS™ prediction will be considered positive if the first two independent runs are positive, a third run is not necessary.
• Because a dose finding assay is not conducted, there is an exception if, in the first run, the S.I. of CD86 is ≥ 150 % at the highest non-cytotoxic concentration only. The run will be considered as not conclusive (NC), and additional concentrations should be tested in additional runs.
• In case a run will be identified as not conclusive, at least two additional runs should be conducted, and a fourth run in case runs 2 and 3 are not concordant. Follow up runs will be considered positive even if only one non-cytotoxic concentration gives a CD86 ≥150 %, since the concentration setting has been adjusted for the test item. The final prediction will be based on the majority result of the four or four individual runs.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

picrylsulfonic acid/2,4,6-trinitro-benzene-sulfonic acid (TNBS) [442E]

Positive control results:

In both experiments, all acceptance criteria including the CD86 stimulation index (S.I.) of the positive and negative controls were met.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

13.4 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

CV70 [442E]

Value:

24.2 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

CV70 [442E]

Value:

28 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not applicable
- The test item showed interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 %) at 50, 100 and 200 μg/mL in the first experiment and at 50 μg/mL in the second experiment. This observation has no impact for the outcome of the study since the concentrations which showed interference did not meet the cytotoxcitiy criterion and were excluded from the assessment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The technical proficiency of the U-SENS™ with the OECD 442E guideline recommended proficiency substances was demonstrated.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative and positive control were met. For details on values please refer to Attachment.

Interpretation of results:

other: The test item is positive for the third key event of the skin sensitisation AOP.

Conclusions:

The test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro skin sensitization test (U-SENS™) according to OECD TG 442E was performed to assess the skin sensitization potential of the test item dissolved in DMSO when administered to U937 cells for 45 ± 3 hours.

The highest test item concentration was 200 μg/mL in accordance with the OECD TG 442E.

For CD86 expression measurement the following concentrations of the test item were tested in the main experiments (U-SENS™):

1, 10, 20, 50, 100 and 200 μg/mL in first experiment

1, 12.5, 15, 20, 25 and 50 μg/mL in the second experiment

In both experiments, all acceptance criteria including the CD86 stimulation index (S.I.) of the positive and negative controls were met.

In the first experiment, cytotoxic effects (cell viability < 70 %) were observed following incubation with the test item starting with a concentration of 50 μg/mL up to the highest tested concentration.

In the second experiment, cytotoxic effects were observed following incubation with the test item starting with 25 μg/mL up to the highest tested concentration.

The CV70 value could be calculated as 28.0 μg/ml in the first experiment and 24.2 μg/mL in the second experiment.

The test item showed interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 %) at 50, 100 and 200 μg/mL in the first experiment and at 50 μg/mL in the second experiment. This observation has no impact for the outcome of the study since the concentrations which showed interference did not meet the cytotoxcitiy criterion and were excluded from the assessment.

In the first experiment, the CD86 stimulation index (S.I.) was higher than 150 % after treatment with a test item concentration starting at 20 μg/mL.

In the second experiment, the CD86 stimulation index (S.I.) was higher than 150 % after treatment in all tested concentrations.

In conclusion, the test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-07-05 to 2021-07-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2020-06-26

Deviations:

yes

Remarks:

Please refer to "Principles of method".

Principles of method if other than guideline:

Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate peptide buffer and acetonitrile (parallel dilution) instead of conducting a serial dilution as stated in the OECD 442C Guideline. This procedure was selected, since this preparation is similar to the preparation of the test item samples and controls. Furthermore, the DPRA proficiency study of the laboratory was conducted under these conditions.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

other:

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
- Preparation of the test chemical solutions
The test item was weighed into volumetric flask and dissolved immediately before testing in acetonitrile, to prepare a 100 mM stock solution.
- Preparation of the positive controls, reference controls and co-elution controls:
The positive control chemical (trans-Cinnamaldehyde) was prepared at a concentration of 100 mM in acetonitrile.
Reference Control A: For the verification of the HPLC system suitability (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=1 with 3-fold injections.
Reference Control B: For the stability of the reference controls over time (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=6.
Reference Control C1: Peptide stability control for the solvent used to dissolve the test item and the positive control (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=3
Co-elution Control: Sample prepared of the respective peptide buffer and the test item or the positive control without peptide. n=1, each
The reference control A, B and C1 samples of both peptides were prepared at a concentration of 500 μM in acetonitrile.
Triplicate solutions each of the positive control and test item stock solutions were diluted with the cysteine peptide stock solution to prepare solutions containing 500 μM cysteine and 5 mM of trans-Cinnamaldehyde or 5 mM of the test item. For the co-elution control, buffer solution was used instead of the cysteine stock solution.
Triplicate solutions each of the positive control and test item stock solutions were diluted with the lysine peptide stock solution so as to prepare solutions containing 500 μM lysine and 25 mM of trans-Cinnamaldehyde or 25 mM of the test item. For the co-elution control, buffer solution was used in place of the lysine stock solution.

INCUBATION
- Incubation conditions
500 μM cysteine and lysine peptide solutions were incubated in glass autosampler vials with 5 mM or 25 mM of the test item, respectively. The reaction solutions were incubated in the dark at 22.5 – 30 ºC for 24 ± 2 hours prior to initiation of the analysis run. The test item and the positive control were analysed in triplicate for both peptides. The appearance of the test item and positive control samples in the HPLC vials was visually inspected and documented after preparation and prior to initiation of the HPLC run.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys
Calibration standards of both peptides were prepared in a solution of 20 % acetonitrile:buffer using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide. The following calibration solutions were prepared from the peptide stock solution of each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A blank of the dilution buffer was also included in the standard calibration curve for both peptides. The blank is 25 % acteonitrile:buffer solution with phosphate buffer (pH 7.5) for the cysteine peptide and with ammonium acetate buffer (pH 10.2) for the lysine peptide without peptide.
For the parameters please refer to “Any other information on materials and methods”.

DATA EVALUATION
- Cys and Lys peptide detection wavelength
220 nm and 258 nm

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Mean Cysteine depletion (%): 72.9 +/- 0.638
Mean Lysine depletion (%): 61.9 +/- 0.636

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1.49 %

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

0.341 %

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not applicable
- Precipitation: A weak precipitate was observed immediately upon addition of the test item solution to the peptide solution. This precipitation was also partially observed after the incubation period of 24 ± 2 hours.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Analysis was performed according to the proficiency study 1841800 conducted at the laboratory.

ACCEPTANCE OF RESULTS:
Please refer to “Any other information on results”.

Acceptance criteria of each peptide

	Peptide	Standard Linearity	Positive control depletion (p.p.)	Reference controls (mean peptide concentration / coefficient of variation)	SD Test item depletion (p.p.)
Acceptance criteria	Cysteine	r2 > 0.99	60.8 - 100 (SD < 14.9)	450 - 550 μM (CV < 15 %)	SD < 14.9
	Lysine	r2 > 0.99	40.2 - 69.0 (SD < 11.6)	450 - 550 μM (CV < 15 %)	SD < 11.6
Achieved results	Cysteine	r2=0.9996	72.9 (SD, 0.638, n=3)	A: 495 μM (CV 1.57 %, n=1) B: 482 μM (CV 1.04 %, n=6) C1: 481 μM (CV 0.611 %, n=3)	SD 0.591 (n=3)
	Lysine	r2=1.000	61.9 (SD, 0.636, n=3)	A: 517 μM (CV 0.742 %, n=1) B: 511 μM (CV 0.656 %, n=6) C1: 512 μM (CV 0.419 %, n=3)	SD 1.28 (n=3)

SD Standard Deviation

p.p. percent points

All analytical acceptance criteria for each peptide run were met.

Interpretation of results:

study cannot be used for classification

Conclusions:

Precipitations were observed upon adding test item to buffer solution. Therefore, the negative result observed cannot be used in an assessment of skin sensitisation potential, as mentioned in the OECD Test Guideline No. 442C, and the DPRA results are considered inconclusive.

Executive summary:

The purpose of this study (based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item.

This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.

The test item was dissolved in acetonitrile and incubated for 24 ± 2 hours in the range between 22.5 and 30 °C.

Solutions of the test item were analysed by the DPRA method in both, cysteine and lysine containing synthetic peptides. There were no co-elution peaks in either the cysteine or lysine assays.

A weak precipitation was observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item. No statement can be made about how much test item remained in the solution to react with the peptide. After an incubation period of 24 ± 2 hours, no precipitate, but rather a type of droplet was observed. Therefore, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence. In the presence of the test item, no to minimal mean depletion of both peptides (0.341 % cysteine depletion and 1.49 % lysine depletion) was observed.

Therefore, the negative result cannot be used in an assessment of skin sensitisation potential, as mentioned in the OECD Test Guideline No. 442C and the DPRA results are considered inconclusive.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-02-16 to 2022-03-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

06 July 2012

Deviations:

yes

Remarks:

Please refer to "Principles of method".

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

Please refer to "Principles of method".

Principles of method if other than guideline:

The relative humidity in the animal room was between approximately 20-65 % instead of 30-70 % for several hours due to a defective humidity sensor. This deviation to the test guideline, however, does not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: No details provided but only animals without any visible signs of illness were used for the study.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15.4-20.2 g
- Housing: group, Makrolon Type II (pre-test) / III (main study) with wire mesh top, on granulated soft wood bedding
- Diet: ad libitum, 2018C Teklad Global 18 % protein rodent diet
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: Not specified.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): approx. 45-65 (20-65 for several hours due to a defective humidity sensor)
- Air changes (per hr): at least 8
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50, and 100 %

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone/olive oil (v/v, 4+1) as vehicle. Vortexing was used to formulate the test item.
- Irritation: Erythema of the scalp and scaly ears were observed.
- Systemic toxicity: Two mice treated with 25 and 50 % test item concentration showed mild, unspecific signs of discomfort, such as partially closed eyes, piloerection, erythema of the scalp, and scaly ears. No clear signs of toxicity were observed, however, and both animals showed a gain in body weight. In a second pre-test was performed using a test item concentration of 100 % in one animal, mild, unspecific signs of discomfort were observed, such as partially closed eyes, piloerection, decreased activity, fluffy cheeks and scaly ears, but no signs of toxicity or loss in body weight.
- Ear thickness measurements: One animal treated with 25 % showed ear swelling of 8.1 % from before first treatment until prior to 3rd treatment and 5.8 % from before first treatment until necropsy. One animal treated with 50 % showed ear swelling of 4.4 % from before first treatment until prior to 3rd treatment and 4.4 % from before first treatment until necropsy. One animal treated with 100 % showed ear swelling of 9.0 % from before first treatment until prior to 3rd treatment and 10.1 % from before first treatment until necropsy.
- Erythema scores: Erythema of the ear skin (Score 1 to 2) was observed in two mice treated with 25 and 50 % test item concentration. One animal treated with 100 % test item had no visible erythema.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item was regarded as a sensitiser in the LLNA if the following criteria were fulfilled:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
Second, that the data were compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and acetone/olive oil (v/v, 4+1) was added (weight per weight). The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion.
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50 % in acetone/olive oil (v/v, 4+1), and 100 % (undiluted test item). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.8 µCi of 3H-methyl thymidine (equivalent to 83.2 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in body weight tables.
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
Within the program, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. One outlier (DPM value determined for animal 19) was detected in the Grubb’s Test but not in the Dean-Dixon-Test, and was therefore not excluded from any calculations. Furthermore, exclusion of the outlier value would not change the overall test result.

Positive control results:

In the positive control experiment the group S.I. values were determined to be 2.4, 3.4 and 9.8 after treatment with 5, 10 and 25 % alpha-Hexylcinnamaldehyde, respectively.

Key result

Parameter:

SI

Value:

4.2

Test group / Remarks:

100 %

Parameter:

SI

Value:

2.8

Test group / Remarks:

50 %

Parameter:

SI

Value:

0.8

Test group / Remarks:

25 %

Parameter:

EC3

Value:

57.1

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Please refer to “Any other information on results” for tables.

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
Please refer to “Any other information on results” for tables.

EC3 CALCULATION
EC3 value were calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c; where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
Please refer to “Any other information on results” for tables.

CLINICAL OBSERVATIONS AND SIGNS OF TOXICITY
No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. The animals showed a very slight to well defined erythema of the ear skin (Score 1 to 2) and scaly ears.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Table 1: Calculation and Results of Individual Data

Vehicle: acetone/olive oil (v/v, 4+1)

Test item concentration			DPM values measured	DPM-BG per animal (2 lymph nodes) a)	S.I.b)
%	Group no.	Animal no.
---	---	BG I	12	---	---
---	---	BG II	19	---	---
0	1	1	1878	1862.5	---
	1	2	2839	2823.5	---
	1	3	3145	3129.5	---
	1	4	2365	2349.5	---
	1	5	2657	2641.5	---
25	2	6	3896	3880.5	1.5
	2	7	1913	1897.5	0.7
	2	8	2034	2018.5	0.8
	2	9	2007	1991.5	0.8
	2	10	1062	1046.5	0.4
50	3	11	7102	7086.5	2.8
	3	12	5150	5134.5	2.0
	3	13	5979	5963.5	2.3
	3	14	8992	8976.5	3.5
	3	15	9030	9014.5	3.5
100	4	16	9905	9889.5	3.9
	4	17	10469	10453.5	4.1
	4	18	10661	10645.5	4.2
	4	19	14273*	14257.5*	5.6
	4	20	8881	8865.5	3.5

BG= Background (1 mL 5 % trichloroacetic acid) in duplicate

1= Control Group for the test item

3-4= Test Groups

S.I.= Stimulation Index

a)= values corrected for mean background value (BGI and BGII).

b)= Stimulation Indices relative to the mean of the control group (Group 1)

*statistical outlier value in the Grubb’s Test

 

Table 2: Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes) a)	SD	S.I.
Vehicle Control Group (acetone/olive oil (v/v, 4+1))	2561.3	482.5	1.0
25 %	2166.9	1038.9	0.8
50 %	7235.1	1750.0	2.8S
100 %	10822.3	2041.3	4.2S

a)=Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

S= statistically significant vs. control group

 

Table 3: Calculation of the EC3 value

	Test item concentration %	S.I.
Test Group 3	50 (a)	2.8 (b)
Test Group 4	100 (c)	4.2 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 57.1% (w/w)

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

Interpretation of results:

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

Conclusions:

The test item was found to be a skin sensitiser and an EC3 value of 57.1 % (w/w) was derived.

Executive summary:

In order to study a possible skin sensitising potential of the test item a local lymph node assay according to OECD TG 429 was performed. Three groups each of five female mice were treated once daily with the test item at concentrations of 25 and 50 % (w/w) in acetone/olive oil (v/v, 4+1), and 100 % (undiluted test item) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments. A control group of five mice was treated with the vehicle (acetone/olive oil (v/v, 4+1)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a b-scintillation counter. All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. The animals showed a very slight to well defined erythema of the ear skin (Score 1 to 2) and scaly ears. Stimulation Indices of 0.8, 2.8, and 4.2 were determined with the test item at concentrations of 25, 50, and 100 %. A clear dose response was observed. The EC3 value calculated was 57.1 % (w/w). The test item was found to be a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

To evaluate the potential of the test item to cause skin sensitisation a step wise approach was used. First an in vitro/ in chemico testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), U937 Line Activation Test (U-SENS™) and ARE-Nrf2 (luciferase test method)) based on the OECD Adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals was performed. The predictions of the conducted tests were not consistent or could not be used for an assessment due to precipitation. Therefore, a local lymph node assay (LLNA) according to OECD TG 429 was conducted. The studies are presented together in a weight of evidence approach. The final result was determined based on the LLNA.

 

OECD 442C (reference 7.4.1-1)

The purpose of this study (based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item. The test item was dissolved in acetonitrile and incubated for 24 ± 2 hours in the range between 22.5 and 30 °C. Solutions of the test item were analysed by the DPRA method in both, cysteine and lysine containing synthetic peptides. There were no co-elution peaks in either the cysteine or lysine assays. A weak precipitation was observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item. No statement can be made about how much test item remained in the solution to react with the peptide. After an incubation period of 24 ± 2 hours, no precipitate, but rather a type of droplet was observed. Therefore, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence. In the presence of the test item, no to minimal mean depletion of both peptides (0.341 % cysteine depletion and 1.49 % lysine depletion) was observed. Therefore, the negative result cannot be used in an assessment of skin sensitisation potential, as mentioned in the OECD Test Guideline No. 442C and the DPRA results are considered inconclusive.

 

OECD 442D (reference 7.4.1-2)

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens; OECD TG 442D) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of the skin sensitization Adverse Outcome Pathway (AOP)) of the test item. To determine the dose range of test item concentrations, two dose finding assays were performed. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 μM (threshold of cytotoxicity: <70 % cell viability) in the first cytotoxicity assay and at a concentration of 178 μM in the second cytotoxicity assay. The CV75 value of the first cytotoxicity test was calculated as 156.1 μM and 47.7 μM in the second one. The test item was tested in 5 independent main experiments, of which 2 main experiments yielded in invalid results, since acceptance criteria regarding cytotoxicity was not met. Only the results of valid main experiments (main experiment 1, 4 and 5) are reported and the original numbering is used. The following concentrations of the test item were tested in the first main experiment: 75.2, 90.2, 108, 130, 156, 187 μM. After treatment with the test item for 48 ± 1 h in the main experiment 1 the luciferase induction is <1.5-fold compared to the solvent control in 5 tested concentrations. Since the test item is considered negative, the acceptance criteria regarding testing at least one cytotoxic concentration is met. To characterize the test item concentrations in more detail, two additional main experiments (main experiment 4 and 5) were conducted with 10 test item concentrations. The following concentrations of the test item were tested: 35.5,42.6, 51.1, 61.3, 73.5, 88.3, 106, 127, 153, 183 μM. After treatment with the test item for 48 ± 1 h the luciferase induction was not above or equal to 1.5-fold compared to the solvent control in all tested concentrations. Since the test item is considered negative, the acceptance criteria regarding testing at least one cytotoxic concentration is met. The LuSens prediction for the test item is considered negative. The acceptance criteria were met: - The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 7.08; ME 4: 4.14; ME 5: 3.57). - The positive control had a relative cell viability ≥70 % as compared to the solvent control (ME 1: 99.43 %; ME 4: 109.16 %; ME 5: 113.23 %). - The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 1.08; ME 4: 1.07; ME 5: 0.30 %), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 0.99; ME 4: 0.66; ME 5: 0.27). - The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 6.6 %; ME 4: 14.5 %; ME 5:10.4 %). - At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability <70 %. In conclusion, the test item did not activate the LuSens cells up to a concentration of 187 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.

 

OECD 442E (reference 7.4.1-3)

This in vitro skin sensitization test (U-SENS™) according to OECD TG 442E was performed to assess the skin sensitization potential of the test item dissolved in DMSO when administered to U937 cells for 45 ± 3 hours. The highest test item concentration was 200 μg/mL in accordance with the OECD TG 442E. For CD86 expression measurement the following concentrations of the test item were tested in the main experiments (U-SENS™): 1, 10, 20, 50, 100 and 200 μg/mL in first experiment; 1, 12.5, 15, 20, 25 and 50 μg/mL in the second experiment. In both experiments, all acceptance criteria including the CD86 stimulation index (S.I.) of the positive and negative controls were met. In the first experiment, cytotoxic effects (cell viability < 70 %) were observed following incubation with the test item starting with a concentration of 50 μg/mL up to the highest tested concentration. In the second experiment, cytotoxic effects were observed following incubation with the test item starting with 25 μg/mL up to the highest tested concentration. The CV70 value could be calculated as 28.0 μg/ml in the first experiment and 24.2 μg/mL in the second experiment. The test item showed interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 %) at 50, 100 and 200 μg/mL in the first experiment and at 50 μg/mL in the second experiment. This observation has no impact for the outcome of the study since the concentrations which showed interference did not meet the cytotoxcitiy criterion and were excluded from the assessment. In the first experiment, the CD86 stimulation index (S.I.) was higher than 150 % after treatment with a test item concentration starting at 20 μg/mL. In the second experiment, the CD86 stimulation index (S.I.) was higher than 150 % after treatment in all tested concentrations. In conclusion, the test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

 

OECD 429 (reference 7.4.1-4)

In order to study a possible skin sensitising potential of the test item a local lymph node assay according to OECD TG 429 was performed. Three groups each of five female mice were treated once daily with the test item at concentrations of 25 and 50 % (w/w) in acetone/olive oil (v/v, 4+1), and 100 % (undiluted test item) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments. A control group of five mice was treated with the vehicle (acetone/olive oil (v/v, 4+1)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a b-scintillation counter. All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. The animals showed a very slight to well defined erythema of the ear skin (Score 1 to 2) and scaly ears. Stimulation Indices of 0.8, 2.8, and 4.2 were determined with the test item at concentrations of 25, 50, and 100 %. A clear dose response was observed. The EC3 value calculated was 57.1 % (w/w). The test item was found to be a skin sensitiser under the test conditions of this study.

 

Conclusion

First an in vitro/ in chemico testing battery was conducted. The negative result of the DPRA (Direct Peptide Reactivity Assay) could not be used in an assessment of skin sensitisation potential as the DPRA results are considered inconclusive due to precipitation. In the ARE-Nrf2 (luciferase test method) study the test item did not activate the LuSens cells up to a concentration of 187 μM. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP. The U937 Line Activation Test (U-SENS™) showed that the test item activated U-937 cells. Therefore, the test item is considered positive for the third key event of the skin sensitisation AOP. The predictions based on the key events of the AOP either cannot be used for an assessment or lead to different estimations of the skin sensitisation potential of the test item. Therefore, the result from the local lymph node assay (LLNA) is considered to be the most reliable and accurate prediction of the test item properties in regard to skin sensitisation. Based on this the test item is determined to be a skin sensitiser.

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.  Based on available data on skin sensitisation, the test item requires classification for causing skin sensitisation (Category 1B, H317: May cause an allergic skin reaction) according to Regulation (EC) No 1272/2008 (CLP).