Registration Dossier

Administrative data

Description of key information

Studies conducted to recognised testing guidelines with GLP certification

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
QSAR: DEREK NEXUS
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Study period:
21 March 2019 - 21 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE

DEREK NEXUS v 6.0.1.

2. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

Chemical name: 5,7-dimethoxy-3-(4-alkyl-benzoyl)-2H-chromen-2-one (major component)
CAS Number Not available
Molecular weight: UVCB (Weight of the major component: 464.59)
Molecular formula C29H36O5

3. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures. LHASA (see Appendix I) has inserted validation comments for the skin sensitization alerts.

4. APPLICABILITY DOMAIN
See QMRF

5. ADEQUACY OF THE RESULT
See QMRF
Qualifier:
no guideline required
Principles of method if other than guideline:
DEREK NEXUS contains 90 alerts for skin sensitisation, together with reasoning rules encoding physicochemical descriptors. In addition to a prediction of skin sensitisation potency for alerting query compounds, Derek evaluates potentially misclassified and unclassified features in cpompounds that do not activate skin sensitsation alerts or examples.
GLP compliance:
no
Remarks:
QSAR data
Justification for non-LLNA method:
QSAR data used as part of an in vitro testing strategy.
Parameter:
other: Structural alerts
Remarks:
structural similarity of 16- 56%
Remarks on result:
other:
Remarks:
11 based on alpha,beta-unsaturated ketone
Parameter:
other: Predicted LLNA EC3
Remarks:
structural similarity of 16- 56%
Remarks on result:
other:
Remarks:
13%, 11 based on alpha,beta-unsaturated ketone
Parameter:
other: Structural alerts
Remarks:
structural similarity of 32- 58%
Remarks on result:
other:
Remarks:
10 based on alpha,beta- unsaturated ester
Parameter:
other: Predicted LLNA EC3
Remarks:
structural similarity of 32-58%
Remarks on result:
other:
Remarks:
22% based on alpha,beta- unsaturated ester

The result as generated by DEREK NEXUS is presented in Appendix 2. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3.

Esacure 3644 is a UVCB of which the major component was assessed for skin sensitizing properties. DEREK NEXUS version 6.0.1 yielded four alerts for the major component of Esacure 3644 for skin sensitization based on the presence of an alpha,beta-unsaturated ketone, an alpha,beta-unsaturated ester, a dihydroxy coumarin, and a vinylic anisole. The dihydroxy coumarin and the vinylic anisole group may have the ability to act as a pre- or prohapten for skin sensitization.

For the alpha,beta-unsaturated ketone alert an EC3 of 13% was predicted based on data from 11 structurally related analogues (structural similarity of 16- 56%). For the alpha,beta-unsaturated ester alert an EC3 of 22% was predicted based on data from 10 structurally related analogues (structural similarity of 32- 58%). For the other two alerts no EC3 could be calculated due to insufficient data on analogues, and consequently the overall, worst case EC3 for Esacure 3644 cannot be determined.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Predicted to be sensitizing to the skin
Conclusions:
In conclusion, the main component of Esacure 3644 is predicted to be sensitizing to the skin (plausible). The overall, worst case EC3 value could not be determined.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
Adopted June, 2018
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Identification: Esacure 3644
Physical Description: Light yellow solid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Details on study design:
TEST SYSTEM
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

RATIONALE
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

CELL CULTURE
Basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).
Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

ENVIRONMENTAL CONDITIONS
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual 95%; based on experiment 2, see deviation), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.8 – 37.8°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

SUBCULTURING
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

EXPERIMENTAL DESIGN
Plating of Cells:
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium.

For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+11 in experiment 1 and P+13 in experiment 2.

Treatment of Cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2. Initially, experiment 1 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 2 valid experiments were performed.

Luciferase Activity Measurement:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

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


VEHICLE
The vehicle of the test item, i.e. dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).

CONTROL ITEMS
Vehicle Control: The vehicle control is the vehicle of the test item.
Positive Control (RS582): The positive control is Ethylene dimethacrylate glycol (Sigma, Zwijdrecht, the Netherlands).

TEST ITEM CHARACTERIZATION
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item(s). The characterization of the test item was conducted in a sponsor or sponsor subcontractor quality environment. A Certificate of Analysis or equivalent document was provided to the Test Facility and is presented in Appendix 7.

RESERVE SAMPLES
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility. The sample will be destroyed after the expiry date.
TEST ITEM INVENTORY AND DISPOSITION
Records of the receipt, distribution, and storage of test item(s) were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
No correction was made for the composition/purity of the test item.
A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 40 mg/ml (yellow solution). The 100-fold dilution in DMEM glutamax of the 40, 20 and 10 mg/ml stock formed a non-homogeneous solution (moderate or heavy precipitate) and was therefore not suitable to test. The 100-fold dilution of the 5 and 2.5 mg/ml DMSO stock formed a homogeneous solution (slight precipitation). The 100-fold dilution of the 1.3 mg/ml and lower DMSO stock in DMEM glutamax formed a homogeneous solution (no precipitation). The concentration of 50 µg/mL (5 mg/ml stock) was selected as highest concentration for the main assay (limit of solubility).
In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at
5 mg/ml (yellow solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.098, 0.049 and
0.024 µg/ml (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.
No precipitation was observed at the start and end of the incubation period in the 96-well plates.
Test item concentrations were used within 2.5 hours after preparation.
Any residual volumes were discarded.

PREPARATION OF THE POSITIVE CONTROL
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.5.1, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate. The formulation of the positive control was used in studies performed concurrently.

PREPARATION OF THE VEHICLE CONTROL
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

BLANK
On each plate three blank wells were tested (no cells and no treatment).

Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.18 and the EC1.5 119 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.43 and the EC1.5 84 µM.
Parameter:
other: luciferase activity induction
Run / experiment:
1
Value:
0.93
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Parameter:
other: luciferase activity induction
Run / experiment:
2
Value:
1.14
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
Esacure 3644 showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 0.93-fold and 1.14-fold in experiment 1 and 2 respectively. Esacure 3644 is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/ml.

Esacure 3644 was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway.  An overview of the viability and luciferase activity induction is summarized in Table 1 and Figure 2-3.  The results of the positive control are summarized in Table 2 and Figure 4-5.  An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3.  The individual raw data are presented in Appendix 3 and Appendix 4.  The historical control data are presented in Appendix 5.

Two independent experiments were performed.  The cells were in these experiments incubated with Esacure 3644 in a concentration range of 0.024 – 50 µg/ml (2-fold dilution steps) for 48 hours ± 1 h.  The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control.  In addition, the viability was assessed with an MTT assay.

Experiment 1

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

•       Esacure 3644 showed no toxicity.  The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.  

•       No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Esacure 3644.  The Imax was 0.93 and therefore no EC1.5 could be calculated.  

•       The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity.  The Imax was 2.18 and the EC1.5 119 µM.  

Experiment 2

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

•       Esacure 3644 showed no toxicity.  The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.  

•       No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Esacure 3644.  The Imax was 1.14 and therefore no EC1.5 could be calculated.  

•       The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity.  The Imax was 2.43 and the EC1.5 84 µM.  

Both tests passed the acceptance criteria:

•       The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.  

•       The EC1.5 of the positive control was within two standard deviations of the historical mean (119 µM and 84 µM in experiment 1 and 2, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (2.18-fold and 2.43-fold in experiment 1 and 2, respectively).

•       Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.0% and 8.1% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, Esacure 3644 is classified as inconclusive under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the ability of Esacure 3644 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

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

Batch M7-238-1807001 of Esacure 3644 was a light yellow solid.  Esacure 3644 was dissolved in dimethyl sulfoxide at 5 mg/ml.  From this stock 11 spike solutions in DMSO were prepared.  The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.024 – 50 µg/ml (2-fold dilution series).  The highest test concentration was considered to be the limit of solubility.  No precipitate was observed at any dose level tested.  Two independent experiments were performed.

Both experiments passed the acceptance criteria:

•       The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.  

•       The EC1.5 of the positive control was within two standard deviations of the historical mean (119 µM and 84 µM in experiment 1 and 2, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (2.18-fold and 2.43-fold in experiment 1 and 2, respectively).

•       Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.0% and 8.1% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

Esacure 3644 showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.  The maximum luciferase activity induction (Imax) was 0.93-fold and 1.14-fold in experiment 1 and 2 respectively.  Esacure 3644 is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/ml.

In conclusion, Esacure 3644 is classified as inconclusive under the experimental conditions described in this report.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 May 2019 - 18 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to
Guideline:
other: EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: Esacure 3644
Physical Description: Light yellow solid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Test Facility test item number: 209751/A
Test item handling: The test item solutions should be prepared in amber glassware or wrapped in aluminum foil to protect them from light
Substance Name: Reaction mass of 5,7-dimethoxy-3-(4-(sec-C10-C13-alkyl)-benzoyl)-2H-chromen-2-one
EC Number: 949-790-7
EC Name: 5,7-dimethoxy-3-[4-(sec-C10-C13-alkyl)-benzoyl]-2H-chromen-2-one
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST SYSTEM
Species: Mouse
Strain: CBA/J
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 11 weeks old) were selected.
Weight at the Initiation of Dosing: 19.8 to 25.6 g.

ENVIRONMENTAL ACCLIMATION
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

HOUSING
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

ENVIRONMENTAL CONDITIONS
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 42 to 58%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

FOOD
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

WATER
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ANIMAL ENRICHMENT
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

VETERINARY CARE
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25 and 60% test item concentration, no signs of systemic toxicity were noted and no irritation was observed. Therefore, a 60% concentration was selected as highest concentration for the main study.
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TEST
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 25% and 60% concentration. The highest concentration was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

INDUCTION - DAYS 1, 2 AND 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

EXCISION OF THE NODES - DAY 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

TISSUE PROCESSING FOR RADIOACTIVITY - DAY 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS - DAY 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).


MORTALITY/MORIBUNDITY CHECKS
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

POSTDOSE OBSERVATIONS
Postdose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

BODY WEIGHTS
Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).

IRRITATION
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing), according to the following numerical scoring system.
Furthermore, a description of all other (local) effects was recorded.
Erythema and eschar formation:

No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

TERMINAL PROCEDURES
No necropsy was performed, since all animals survived until the end of the observation period.

Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
Parameter:
SI
Value:
1.7
Test group / Remarks:
10% test concentration
Parameter:
SI
Value:
1.4
Test group / Remarks:
25% test concentration
Parameter:
SI
Value:
1.4
Test group / Remarks:
60% test concentration
Cellular proliferation data / Observations:
Skin reactions/ irritation
The very slight erythema of the ears as shown by animals treated at 60% on Days 2 and/or 3 was considered not to have a toxicologically significant effect on the activity of the nodes.
White test item remnants were present on the dorsal surface of the ears of test item treated animals between Days 1 and 5, which did not hamper scoring of the skin reactions.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area
The auricular lymph nodes of the control animals and the animals treated at 60% were considered normal in size and the auricular lymph nodes of the animals treated at 10% and 25% were considered to be slightly enlarged compared to normal. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 887, 703 and 731 DPM, respectively. The mean DPM/animal value for the vehicle control group was 508 DPM. The SI values calculated for the test item concentrations 10, 25 and 60% were 1.7, 1.4 and 1.4, respectively.
Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, Esacure 3644 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 60%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 3).

Based on these results, Esacure 3644 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Executive summary:

Test item concentrations selected for the main study were based on the results of a pre-screen test.  At a 25 and 60% test item concentration, no signs of systemic toxicity were noted and no irritation was observed.  Therefore, a 60% concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 60% w/w on three consecutive days, by open application on the ears.  Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)).  Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.  After precipitating the DNA of the lymph node cells, radioactivity measurements were performed.  The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The auricular lymph nodes of the control animals and the animals treated at 60% were considered normal in size and the auricular lymph nodes of the animals treated at 10% and 25% were considered to be slightly enlarged compared to normal.  No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 887, 703 and 731 DPM, respectively.  The mean DPM/animal value for the vehicle control group was 508 DPM.  The SI values calculated for the test item concentrations 10, 25 and 60% were 1.7, 1.4 and 1.4, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, Esacure 3644 was considered not to be a skin sensitizer.  It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 60%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, Esacure 3644 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.  The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint:
skin sensitisation, other
Remarks:
Weight of Evidence report
Type of information:
other: Weight of Evidence approach
Adequacy of study:
supporting study
Study period:
22 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
A DEREK assessment and a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A DEREK assessment and a KeratinoSensTM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.
GLP compliance:
no
Remarks:
DEREK assessment and a KeratinoSensTM assay were performed under GLP conditions.
Type of study:
other: Weight of evidence approach report using DEREK assessment and a KeratinoSensTM assay
Specific details on test material used for the study:
Identification: Esacure 3644
Substance name: Reaction mass of 5,7-dimethoxy-3-(4-(sec-C10-C13-alkyl)-benzoyl)-2H-chromen-2-one
CAS number: Not available
EC number: 949-790-7
EC name: 5,7-dimethoxy-3-[4-(sec-C10-C13-alkyl)-benzoyl]-2H-chromen-2-one Purity UVCB
Molecular weight: UVCB (Weight of the major component: 464.59)

No data were available that would preclude performance of the studies to determine the potential for skin sensitization. Therefore, STEP 1 studies were performed, i.e. a DEREK assessment (study no 20186176) and a KeratinoSensTM assay (study no 20186178).

Results of studies performed

DEREK NEXUS version 6.0.1 yielded four alerts for the major component of Esacure 3644 for skin sensitization based on the presence of an alpha,beta-unsaturated ketone, an alpha,beta-unsaturated ester, a dihydroxy coumarin, and a vinylic anisole. The dihydroxy coumarin and the vinylic anisole group may have the ability to act as a pre- or prohapten for skin sensitization.For two of the four alerts no EC3 could be calculated due to insufficient data on analogues, and consequently the overall, worst case EC3 for Esacure 3644 cannot be determined. In conclusion, the main component of Esacure 3644 is predicted to be sensitizing to the skin (plausible). The overall, worst case EC3 value could not be determined.

A valid KeratinoSensTM assay was performed according to OECD 442D and GLP. For the KeratinoSensTM assay Esacure 3644 was dissolved in dimethyl sulfoxide to a final concentration of 5 mg/mL (yellow solution). Test concentrations of 0.024 – 50 μg/mL were used in the experiments. The highest test concentration was considered to be the limit of solubility. Two independent experiments were performed. No precipitation and cytotoxicity was observed at all dose levels tested. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 0.93-fold and 1.14-fold in experiment 1 and 2 respectively. Esacure 3644 is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/ml.

Interpretation of results:
study cannot be used for classification
Conclusions:
Since the current data set is not adequate for classification and risk assessment on the endpoint skin sensitization and further in vitro testing would not lead to a conclusion, it is considered justified to perform an in vivo study to assess the skin sensitizing properties of Esacure 3644.
Executive summary:

The DEREK assessment predicted Esacure 3644 to be a skin sensitizer based on four different groups present. However, no overall worst case EC3 could be determined. The KeratinoSensTM assay showed no activation of keratinocytes up to and including the highest concentration tested (limit of solubility). As the top concentration was too low to determine a reliable result in the KeratinoSensTM assay the outcome was regarded as inconclusive.

As DEREK predicted that metabolism might play a role in the sensitizing ability of Esacure 3644, and the KeratinoSensTM assay has only limited metabolic capacity, the absence of any induction might also be false. Based on these results additional testing is needed however performing a U-SENSTM assay as a next step would not lead to a definitive conclusion on the skin sensitizing properties of Esacure 3644. In case of a positive outcome the substance is regarded as a sensitizer and potency would need to be determined in vivo. As metabolism may play a role in skin sensitization of Esacure 3644, a negative outcome of the U-SENSTM assay would still require an in vivo assay to be performed, since the U-SENSTM assay only has limited metabolic capacity and may be false negative.

Since the current data set is not adequate for classification and risk assessment on the endpoint skin sensitization and further in vitro testing would not lead to a conclusion, it is considered justified to perform an in vivo study to assess the skin sensitizing properties of Esacure 3644.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Since the current data set is not adequate for classification and risk assessment on the endpoint skin sensitization and further in vitro testing would not lead to a conclusion, it is considered justified to perform an in vivo study to assess the skin sensitizing properties of Esacure 3644.