4-(3-Butenyl)-4''-ethyl-2'-fluor-1,1':4',1''-terphenyl

Endpoint summary

Description of key information

A solubility assessment showed precipitation upon adding test item to buffer solution used for the OECD 442C assay. 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 66.7 μM (limited by observed precipitations) 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).

Due to the conflicting in vitro results, a further in vivo assay according to OECD 429 was conducted. The test item was not a skin sensitiser under the test conditions of this study. (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 vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-02-14 to 2022-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 429

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 45-65% for severaours on several days during the first few days of the acclimation period of the animals used in the main experiment.

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: 16.0 - 20.3 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 a 50% solution in MEK. Vortexing was used to formulate the test item.
- Irritation: At the tested concentrations the animals did not show any signs of local skin irritation
- Systemic toxicity: Both animals showed transient, mild and unspecific signs of discomfort: The animal treated with 50% of the test item showed partially closed eyes (transiently at the post-dose observation time after the 2nd and 3rd application). Additionally, the animal treated with 50% test item concentration showed piloerection and decreased activity at the post-dose observation time on day 3. Both animals showed a normal gain in body weight over the course of the study.
- Ear thickness measurements: No findings
- Erythema scores: No irritation observed

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 MEK 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 10, 25, and 50% in MEK. 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 19.8 µCi of 3H-methyl thymidine (equivalent to 79.3 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:

1.11

Test group / Remarks:

50 %

Parameter:

SI

Value:

1.64

Test group / Remarks:

25 %

Parameter:

SI

Value:

1.19

Test group / Remarks:

10 %

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 symptoms of local skin irritation at the ears of the animals were observed during the study period. The animals treated with a test item concentration of 50% showed mild and unspecific signs of discomfort, namely, partially closed eyes and piloerection at the post-dose observation time points on days 2 and 3, possibly due to substance residuals on the ears. Animals treated with 10 and 25%% test item concentration did not show any clinical signs.

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.

 Calculation and Results of Individual Data

Vehicle: MEK

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	12	---	---	---	---
---	BG II	26	---	---	---	---
0	1	5480	5461	8	682.6	1.00
10	2	6540	6521	8	815.1	1.19
25	3	8957	8938	8	1117.2	1.64
50	4	6100	6081	8	760.1	1.11

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item formulated in MEK (methyl ethyl ketone) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/w). The highest concentration tested was the highest concentration that could technically be achieved.

The animals did not show any signs of local skin irritation during the course of the study and no cases of mortality were observed. The animals treated with a test item concentration of 50% showed mild and unspecific signs of discomfort, such as partially closed eyes and piloerection, directly after the 2nd and 3rd application only. Animals treated with 10 and 25%% test item concentration did not show any clinical signs.

In this study Stimulation Indices (S.I.) of 1.19, 1.64, and 1.11 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in MEK, respectively.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-11-03 to 2022-03-11

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


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
Six test item concentrations were tested in the main experiments.
Since no CV75 could be determined, 100 μM was used as highest test item concentration and five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2 in the first main experiment.
Due to observed precipitations the top dose for the second main experiment was 80 μM and five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2.
first main experiment: 40.2, 48.2, 57.9, 69.4, 83.3, 100 μM
main experiment 2: 32.2, 38.6, 46.3, 55.6, 66.7, 80.0 μM

- 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 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:

80 µM

Cell viability:

precipitation

Group:

test chemical

Run / experiment:

other: first cytotoxicity test

Parameter:

CV75 [442D and 442E]

Value:

69.4 µM

Cell viability:

precipitation

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:
The acceptance criteria were met:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 33.07; ME 2: 7.76) and statistically significant.
- The positive control had a relative cell viability ≥70% as compared to the solvent control (ME 1: 100.20%; ME 2: 95.48%).
- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 1.12; ME 2: 0.95), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 1.48; ME 2: 1.2).
- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 7.8%; ME 2: 7.7%).
- 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% or showed precipitates.

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 66.7 μM (limited by observed precipitations) 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) 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 material.

In the dose finding assay, cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (2000 μM). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Precipitations were observed starting with the test item concentration of 125 μM up to the highest test item concentration. In this case, the OECD 442D guideline recommends testing a test item concentration of the highest soluble or stably dispersible test item concentration. Five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2.

The test item was tested in 2 independent main experiments.

The following concentrations of the test item were tested in the first main experiment:

40.2, 48.2, 57.9, 69.4, 83.3, 100 μM

Due to observed precipitations the test item concentrations for the second main experiment were adjusted to:

32.2, 38.6, 46.3, 55.6, 66.7, 80.0 μM

After treatment with the test item for 48 ± 1 h the luciferase induction is <1.5-fold compared to the solvent control in all tested concentrations without precipitations. Since these conditions are met in 2 of 2 main experiments, the LuSens prediction 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: 33.07; ME 2: 7.76) and statistically significant.

- The positive control had a relative cell viability ≥70% as compared to the solvent control (ME 1: 100.20%; ME 2: 95.48%).

- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 1.12; ME 2: 0.95), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 1.48; ME 2: 1.2).

- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 7.8%; ME 2: 7.7%).

- 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% or showed precipitates.

In conclusion, the test item did not activate the LuSens cells up to a concentration of 66.7 μM (limited by observed precipitations) 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 3

Endpoint:

skin sensitisation: in vitro

Study period:

2022-01-13

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Qualifier:

no guideline available

Guideline:

other: Pre Test for compound solubility

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Pre Test for solubility

Details of test system:

other: solubility test

Details on the study design:

PREPARATION OF TEST SOLUTIONS
For the solubility, a test item concentration of 100 mM was prepared in the solvent acetonitrile. Therefore, the test item was weight into a glass test tube and the appropriate volume of the solvent was added.
The further dilution in the phosphate buffer occurred by mixing 100 μL of the 100 mM solution with 1500 μl phosphate buffer pH 7.5 and 400 μL acetonitrile.
The further dilution in the ammonium acetate buffer occurred by mixing 500 μL of the 100 mM solution were added to 1500 μl ammonium acetate buffer pH 10.2.

Vehicle / solvent control:

other: acetonitrile, methanol, ethanol, acetone, deionized water, isopropanol, water:acetonitrile (1:2, v/v), acetone:acetonitrile (1:2, v/v)

Outcome of the prediction model:

other: not soluble

Interpretation of results:

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

Conclusions:

The test item is considered not suitable for the DPRA Assay.

Executive summary:

This study was performed to assess the solubility of the test material in different solvents to conduct a DPRA.

An appropriate solvent for the DPRA test should dissolve the test item completely. The solvents acetonitrile, methanol, ethanol, acetone, deionized water, isopropanol, water:acetonitrile (1:2, v/v), acetone:acetonitrile (1:2, v/v) were shown to be suitable for the DPRA Assay.

To test the solubility, a test item concentration of 100 mM was prepared in the solvent acetonitrile, resulting in a complete dissolved test item. After adding the test item-acetonitrile solution to the phosphate buffer (pH 7.5) or the ammonium acetate buffer (pH 10.2), the formulations were cloudy and showed white drops on the surface.

Precipitation was observed upon addition of the test item solution to both phosphate buffers (pH 7.5 + 10.2), due to low aqueous solubility of the test item.

The test item could be dissolved complete in acetonitrile but resulted in a cloudy formulation after diluting the test item stock solution in peptide buffer. Therefore, the test item is considered not suitable for the DPRA Assay.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-10-27 to 2022-02-01

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 in the first experiment and 1, 10, 15, 20, 35 and 50 μg/mL in the second experiment.
- 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.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD86>200 [442E]

Value:

196 %

At concentration:

20 other: µg/mL

Cell viability:

precipitation @ 50 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD86>200 [442E]

Value:

291.1 %

At concentration:

35 other: µg/mL

Cell viability:

precipitation @ 50 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:
In all experiments, cytotoxic effects (cell viability <70%) and morphological alternations were not observed following incubation with the test material up to the highest tested concentration. Due to the lack of cytotoxicity, a CV70 value could not be calculated.


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 material 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™) was performed to assess the skin sensitization potential of the test material dissolved in DMSO when administered to U937 cells for 45 ± 3 h.

This U-SENS™ test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), and ARE-Nrf2 (luciferase test method)) based on the OECD Adverse Outcome Pathway (AOP) for the assessment of the skin sensitisation potential of chemicals.

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

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

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

1, 10, 15, 20, 35 and 50 μg/mL in the second experiment.

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

In all experiments, cytotoxic effects (cell viability <70%) and morphological alternations were not observed following incubation with the test material up to the highest tested concentration. Due to the lack of cytotoxicity, a CV70 value could not be calculated.

In the first experiment, the CD86 stimulation index (S.I.) was higher than 150% after treatment with the test item concentration of 20 μg/mL. The higher tested concentrations showed a CD86 stimulation index (S.I.) higher to 150% as well but were excluded due to observed precipitations.

In the second experiment, the CD86 stimulation index (S.I.) was higher than 150% after treatment with the test item between 1 μg/mL and 35 μg/mL. The highest test concentration of 50 μg/mL showed precipitation and was excluded from the evaluation.

In conclusion, the test material 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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 and conflicting results. 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. An initial solubility assessment showed precipitation upon adding test item to buffer solution used for the assay. Therefore, the study is technicall not possible.

 

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. The test item did not activate the LuSens cells up to a concentration of 66.7 μM (limited by observed precipitations) 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.The test item activated U-937 cells and 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. In the study test item concentrations of 10, 25, and 50% (w/w) were applied. The highest concentration tested was the highest concentration that could technically be achieved. The animals did not show any signs of local skin irritation during the course of the study and no cases of mortality were observed. The animals treated with a test item concentration of 50% showed mild and unspecific signs of discomfort, such as partially closed eyes and piloerection, directly after the 2nd and 3rd application only. Animals treated with 10 and 25%% test item concentration did not show any clinical signs. 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. In this study Stimulation Indices (S.I.) of 1.19, 1.64, and 1.11 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in MEK, respectively. Based on these data it can be concluded that the test item was not a skin sensitiser under the test conditions of this study.

Conclusion

First an in vitro/ in chemico testing battery was conducted. The DPRA (Direct Peptide Reactivity Assay) could not be used due to technical limitations caused by limited solubilty of the test material. 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 not a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.  Based on available data on skin sensitisation, the test item does not require classification for causing skin sensitisation according to Regulation (EC) No 1272/2008 (CLP).