Diethyl ethoxymethylenemalonate

Endpoint summary

Description of key information

In silico, in chemico and in vitro data are used in a weight-of-evidence approach (see attached document). In addition, the Local Lymph Node Assay (LLNA) is added as key study.

 

- In silico: A Derek Nexus assessment yielded an alert for skin sensitisation based on the presence of enol ether (Alert 425) and alpha,beta-unsaturated ester or precursor (Alert 481) and predicted an EC3 of 21% and 0.58% respectively (De Vlieger, 2017).

- In chemico (OECD 442C): The test item was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model (Rijk, 2018).

- In vitro (OECD 442D): In the KeratinoSens^TM assay the test item was classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described (Westerink, 2018).

- In vitro (OECD 442E): The test item was classified as positive in the U-sens^TM assay since positive results (>150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control (Eurlings, 2018).

In conclusion, taking into account (1) the positive in silico, in chemico and in vitro (i.e. in the U-SENS^TM assay) study results and (2) the fact that it is not possible to determine the potency (Cat. 1A or 1B) based on non-animal testing approaches, as required in Annex VII, section 8.3, additional in vivo testing (Local Lymph

Node Assay in mice according to OECD 429) had to be performed to assess the potency of the test item.

 

- In vivo (OECD 429): Based on the results of a local Lymph Node Assay in female CBA/J mice the test item was regarded as a skin sensitizer category 1A (van Sas, 2019).

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:

key study

Study period:

2018-07-25 to 2018-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Union No. L142, May 2008, including most recent amendments

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: JC170405
- Expiration date of the lot/batch: 2018-10-27 (retest date)
- Purity test date: 2018-08-21
- Purity: 99.4% (GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

OTHER SPECIFICS:
correction factor : 1.00

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: 20 female (nulliparous and non-pregnant) mice, CBA/J strain, inbred, SPF-Quality from Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: within +/- 20% of the sex mean
- Housing: group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material . Paper and shelters were supplied as cage-enrichment. On day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days before the start of treatment, under laboratory conditions. Health inspection at least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.
- Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 48 to 70%.

Vehicle:

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

Concentration:

Pre-Screen Test: 0.5, 1, 2, 5, 50 and 100% (w/w)
Main Study: 0.1, 0.2 and 0.5% (w/w)

No. of animals per dose:

5 females per group; 4 groups

Details on study design:

RANGE FINDING TESTS:
- 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 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
- The test system, procedures and techniques were identical as those used in the main study except that the animals were aproximately 10-12 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals 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.
- Based on the results of the initially treated animals, eight additional animals (two animals per test item concentrations) were treated in a similar manner with four lower concentrations (0.5%, 1%, 2% and 5%) at a later stage.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT - INDUCTION days 1, 2, 3
- 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 vehicle.
- The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day for three consecutive days. 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.
- Criteria used to consider a positive response: a Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated 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 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, including all amendments.The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation.
Classification of results: UN-GHS 2007; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skin reaction
EC3 value ≤ 2%: sub-category 1A
EC3 value ≥ 2%: sub-category 1B

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
- After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. 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 approximately 3 mL 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) 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 as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter. 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).

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item preparations (w/w) were prepared within 4 hours prior to each dosing.
- No adjustment was made for specific gravity of the vehicle.
- Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.

Positive control substance(s):

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

Positive control results:

The SI values calculated for the item concentrations 5, 10 and 25% were 1.1, 2.0 and 5.5 respectively. An EC3 value of 14.3% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 19.2%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Value:

2.5

Variability:

± 0.3

Test group / Remarks:

based on 5 animals of the 0.1% group

Parameter:

SI

Value:

3.6

Variability:

± 0.6

Test group / Remarks:

based on 5 animals of the 0.2% group

Parameter:

SI

Value:

5

Variability:

± 0.7

Test group / Remarks:

based on 5 animals of the 0.5% group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 651 ± 98
2% w/w group: mean DPM ± SEM: 1633 ± 166
25% w/w group: mean DPM ± SEM: 2352 ± 378
50% w/w group: mean DPM ± SEM: 3242 ± 428
SEM = Standard Error of the Mean

DETAILS ON STIMULATION INDEX CALCULATION: The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group.

EC3 CALCULATION: An EC3 value of 0.15% was calculated

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: No irritation of the ears was observed in any of the animals examined.
- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopy of the auricular lymph nodes and surrounding area: All auricular lymph nodes of the animals of the control group, two animals treated at 0.2% and all animals treated at 0.5% were considered normal in size. The auricular lymph nodes of all animals treated at 0.1% and three animals treated at 0.2% were considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Pre-screen test

At a 100% test item concentration animals were killed in extremis on Day 3.

At 50%, 5%, 2% and 1% test item concentrations signs of systemic toxicity were noted and/or variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values.

At a 0.5% test item concentration no signs of systemic toxicity were noted and no irritation was observed and variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values, except for one ear of one animal on Day 3, which was 27% thickened (22% the other ear on Day 3, both ears 11% on Day 6), this was considered to be an incidental finding as ear thickness was not increased for the other ear and both ears of the other animal and was well below 25% on Day 6.

Based on these results, the highest test item concentration selected for the main study was a 0.5% test item concentration.

Interpretation of results:

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

Conclusions:

These results show that the test item elicits a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 0.15% was calculated.
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:
- according to the recommendations made in the test guidelines (including all amendments), JNJ-39005525-AAA (EMME) would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), JNJ-39005525-AAA (EMME) should be classified as skin sensitizer (Category 1A).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), JNJ-39005525-AAA (EMME) should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-09-13 to 2017-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: JC170405
- Expiration date of the lot/batch: 2018-04-12
- Purity (GC): 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the cysteine and lysine reactivity assay 37.72 mg of the test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1744 µL ACN (Fisher Chemicals, Loughborough, England) to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved.

OTHER SPECIFICS: no correction (correction factor =1.00) was made for the purity/composition

Details on the study design:

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

TEST SYSTEM
- Test system: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC, and 775.9 g/mol for SPCL.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Batch SPCC: 111016HS_MHe_W0417
- Batch SPCL: 220114HSDW_W0417
- Storage: The peptides were stored in the freezer (≤ 15°C) for a maximum of 6 months.

EXPERIMENTAL DESIGN
TEST ITEM PREPARATION
see details under "Specific details on test material used for the study"

PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- SPCC stock solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC reference control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.
- SPCC calibration curve: A SPCC calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- SPCL stock solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL reference control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.
- SPCL calibration curve: A SPCL calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

SAMPLE INCUBATIONS
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 22.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 11 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation.

HPLC-PDA Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following system:
System 1 (used for Cysteine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
- LC Column oven 300 (Thermo Scientific)
- Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
- Column Oven #151006 (Grace, Worms, Germany)
- Surveyor PDA detector (Thermo Scientific)

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have an r²>0.99.
- The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
- The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
- The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
- The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
- The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.
All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not have been exactly reproduced from the individual data presented.

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
% Peptide Depletion = [ 1 - (Peptide Peak Area in Replicate injection at 220 nm / Mean Peptide Peak Area in Reference Controls at 220 nm) ] x 100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Positive control results:

Cysteine reactivity assay:
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde, calculated versus the mean SPCC peak area of Reference Controls C, was 71.3% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). This was just below the lower limit of the historical positive control data range.

Lysine reactivity assay:
The Percent SPCL Depletion for the positive control cinnamic aldehyde, calculated versus the mean SPCL peak area of Reference Controls C, was 41.1% ± 1.9%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%). This was just below the lower limit of the historical positive control data range.

Run / experiment:

other: Run 1 / mean of SPCC and SPCL depletion

Parameter:

other: Mean of SPCC and SPCL depletion (%)

Value:

58.4

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Positive: High reactivity class

Run / experiment:

other: Run 1 / mean of 3 replicates

Parameter:

other: % SPCC depletion

Value:

53.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Run 1 / mean of 3 replicates

Parameter:

other: % SPCL depletion

Value:

63.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The DPRA assay was successfully validated at the laboratory and can be used to support the discrimination between sensitisers and non-sensitisers.

ACCEPTANCE OF RESULTS - Cysteine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.993 was within the acceptance criteria (r²>0.99) (SPCC standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.512 ± 0.006 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.525 ± 0.012 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A and C were both within the acceptance criteria which confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 2.7% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 71.3% was within the acceptance range of 60.8% to100%
- SD of peptide depletion cinnamic aldehyde: 0.9% was below the maximum (SD <14.9%)

ACCEPTANCE OF RESULTS - Lysine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.997 was within the acceptance criteria (r²>0.99) (SPCL standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.503 ± 0.007 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.506 ± 0.008 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A and C were both within the acceptance criteria which confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 1.0% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 41.1% was within the acceptance range of 40.2% to 69.0%
- SD of peptide depletion cinnamic aldehyde: 1.9% was below the maximum (SD <11.6%)

Solubility assessment:

At a concentration of 100 mM, the test item was soluble in ACN. Therefore this solvent was used to dissolve the test item in this DPRA study and no other solvents were tested.

Results cysteine reactivity assay for the test item:

Preparation of a 100 mM stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected.  No precipitate was observed in any of the samples.

In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the test item samples, the mean SPCC A220/A258 area ratio was 19.85. This was outside the 16.11 -19.69 range (mean A220/A258 ratio ± 10% range of Reference controls A, B and C). However, since the test item displayed high reactivity towards SPCC, accurate calculation of the peak purity was not possible due to the low SPCC signal at 258 nm.  Overall, it can be concluded that the test item did not co-elute with SPCC.

Results lysine reactivity assay for the test item:

Preparation of a 100 mM stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected.  No precipitate was observed in any of the samples.

In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the test item samples, the mean SPCL A220/A258 area ratio was 14.23 which was within the 12.50 -15.27 range (mean A220/A258 ratio ± 10% range of Reference controls A, B and C). This again indicated that there was no co-elution of the test item with SPCL.

SPCC and SPCL depletion, DPRA prediction and reactivity classification:

SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
53.1%	± 1.6%	63.7%	± 1.0%	58.4%	Positive: High reactivity

SD = Standard Deviation                                 

Historical control data for DPRA studies

	Positive control - Cinnamic aldehyde
	SPCC depletion	SPCL depletion
Range	71.8 - 78.1%	43.5 - 65.2%
Mean	74.8%	59.1%
SD	1.7%	5.2%
n	31	31

SD = Standard Deviation, n = Number of observations

The above mentioned historical control data were collected over the period of January 2017 to August 2017.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.
The test item was positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-10-13 to 2017-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: JC170405
- Expiration date of the lot/batch: 2018-04-12 (retest date)
- Purity (GC): 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM. This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series).

OTHER SPECIFICS: No correction was made for the purity/composition of the test item as the correction factor is 1.

Details on the study design:

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

CONTROLS
- solvent control: 1% DMSO in exposure medium
- positive control: Ethylene dimethacrylate glycol

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- A solubility test with dimethyl sulfoxide (DMSO) was performed as described in Specific details on test material used for this study
- In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). 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 3 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, for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, 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.

SOLVENT CONTROL
The solvent 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).

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 KeratinoSensTM cell line). The KeratinoSensTM cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells were 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 of 25 passages from the stock (p+25) and are employed for routine testing using the appropriate maintenance medium. Once a year the cell line is checked for infection with a mycoplasma detection test.

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

ENVIRONMENTAL CONDITIONS
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 – 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.1 – 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. The temporary deviations, of less than an hour, 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.

EXPERIMENTAL DESIGN
- In total 2 valid experiments were performed. Initially, experiment 2 was rejected due to unexpected lower viability in the positive control (not reported) and therefore this part of the study (including the test item, positive and negative control) was repeated.
- Subculturing: Cells were subcultured upon reaching 80-90% confluency in maintenance medium. 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).
- 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, on ewhite 96-well plate (three replicates per concentration) was used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator 24 ± 1 hours. The passage number used was p+15 in experiment 1 and p+8 in experiment 2.
- Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL exposure 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 then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. Initially, experiment 2 was rejected due to unexpected lower viability in the positive control (not reported) and therefore this part of the study (including the test item, positive and negative control) was repeated.
- Luciferase activity measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together at room temperature. Prior to addition to the cells the Steady-Glo Luciferase substrate was mixed 1:1 with exposure medium. The assay plates were removed from the incubator and the medium was removed. Then 100 μL of PBS was added to rinse the cells. After removing the PBS, 200 µL of the Steady-Glo Luciferase substrate solution 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) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the average induction in the three replicates for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
The coefficient of variation is calculated with the following formula:
Coefficient of Variation = [(Standard deviation / Mean luminescence reading) x 100%]

DATA EVALUATION AND STATISTICAL PROCEDURES
The following parameters were calculated in the KeratinoSensTM test method:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained.
- The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
Imax, EC1.5, IC50 and IC30 were calculated based on the equations stated in the OECD Guideline 442D.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration. ToxRat Professional will be used for statistical analysis of the data.
Additional points for data analysis:
- A graph was produced to help visually check the data. If no clear dose response curve was observed, or if the dose-response curve obtained was biphasic (i.e. crossing the threshold of 1.5 twice), the experiment was repeated to verify whether this was specific to the test chemical or due to an experimental artefact. In case the biphasic response was reproducible in an independent experiment, the lower EC1.5 value (the concentration when the threshold of 1.5 is crossed the first time) should be reported.
- In the rare cases where a statistically non-significant induction above 1.5 fold was observed followed by a higher concentration with a statistically significant induction, results from this repetition were only considered as valid and positive if the statistically significant induction above the threshold of 1.5 was obtained for a non-cytotoxic concentration.
- Finally, for test chemicals generating a 1.5 fold or higher induction already at the lowest test concentration of for example 0.977 μM, the EC1.5 value of <0.977 was set based on visual inspection of the dose-response curve.

DATA INTERPRETATION
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM.
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations < 1000 µM should be considered as inconclusive.

Positive control results:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (108 µM and 58 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.25-fold and 2.65-fold in experiment 1 and 2, respectively).

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.02

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No EC1.5, IC30 and IC50 values could be calculated

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No EC1.5, IC30 and IC50 values could be calculated

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The KeratinoSensTM assay was successfully implemented and validated, and lab proficiency has been shown by obtaining the expected KeratinoSensTM prediction for the 10 proficiency chemicals that are described in the OECD 442D guideline.

ACCEPTANCE OF RESULTS:
All tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 µM (108 µM and 58 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.25-fold and 2.65-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (3.0% and 10.7% in experiment 1 and 2, respectively).

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

Test item results

Experiment 1

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

- The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no 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 the test item. The Imax was 1.02 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 2.25 and the EC1.5 108 μM.

Experiment 2

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

- The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no 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 the test item. The Imax was 1.25 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 2.65 and the EC1.5 58 μM.

Historical Control Data for the KeratinoSensTM Studies:

	Positive control
	EC1.5 (µM)	Imax (µM)
Range	6.3 - 119.2	1.8 - 52.7
Mean	42.9	4.8
SD	28.3	6.8
n	81	77

SD = Standard Deviation; n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2016 to July 2017.

Interpretation of results:

GHS criteria not met

Remarks:

since negative results (<1.5-fold induction) were observed at test concentrations ≥ 1000 μM

Conclusions:

In conclusion, the test item is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2018-03-06 to 2018-03-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E: In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-sens (TM))

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: JC170405
- Expiration date of the lot/batch: 2018-04-12
- Purity (GC): 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed. The test item was either dissolved or suspended in complete medium and dimehtyl sulfoxide (DMSO) to a final concentration of 50 mg/mL. The test item formed a non-homogenous suspension in complete medium at 50 mg/mL. In DMSO the test item formed clear solution at 50 mg/mL. DMSO was selected as solvent for the main assay.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL.
- Final dilution of a dissolved solid, stock liquid or gel: The stock was diluted to final test concentrations of 200, 100, 50, 20, 10 and 1 μg/mL (experiment 1), 200, 180, 140 and 100 μg/mL (experiment 2) and 200, 140, 100, 80, 50, 20, 10 and 1 μg/mL (experiment 3) in the 96-well plate (final concentration DMSO of 0.4%).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution in dimethyl sulfoxide

OTHER SPECIFICS: No correction was made for the purity of the test item as the correction factor is 1.00.

Details on the study design:

CONTROLS:
- Untreated control (RPMI)
- Vehicle control: 0.4% DMSO in RPMI
- Negative control: lactic acid
- Positive control: 2, 4, 6-trinitrobenzenesulfonic acid

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- A solubility test with dimethyl sulfoxide (DMSO) was performed as described in Specific details on test material used for this study.
- In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL. The stock was diluted to final test concentrations of 200, 100, 50, 20, 10 and 1 μg/mL (experiment 1), 200, 180, 140 and 100 μg/mL (experiment 2) and 200, 140, 100, 80, 50, 20, 10 and 1 μg/mL (experiment 3) in the 96-well plate (final concentration DMSO of 0.4%). No precipitation was observed at the end of the incubation period in the 96-well plates.
- Test item concentrations were used within 3 hours after preparation. Any residual volumes were discarded.

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

PREPARATION OF THE POSITIVE CONTROL
2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599) was provided as 1 M solution. On the treatment day a 10 mg/mL solution was prepared in RPMI. This solution was diluted 1:100 with RPMI in order to obtain a 100 μg/mL working solution (final dose level 50 μg/mL).

VEHICLE CONTROL
The vehicle control was 0.4% DMSO in complete medium.

TEST SYSTEM
- Test system: U937 human monocytes
- Justification: inducible CD86-expressing cells
- Source: ATCC (American Type Culture Collection, Virginia, USA); ATCC no.: CRL-1593.2TM
- Stock cultures of these cells are stored in liquid nitrogen (-196°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test.

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

ENVIRONMENTAL CONDITIONS
All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 70 – 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range
35.7 – 36.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations, of less than an hour, from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

EXPERIMENTAL DESIGN
- Three experiments were conducted per test item to demonstrate reproducibility of the results and conclusion.
- Plating of cells: Cultures were initiated in 96-well plates using 100 μL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding.
Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.
- Treatment of cells: Cells were treated for 45 ± 3 hours with the selected doses or controls (100 μL). The test item was evaluated up to 200 μg/mL in the first experiment using six doses: 1.0, 10, 20, 50, 100 and 200 μg/mL. In the second and third experiment cells were treated with four and eight selected doses of test item, respectively. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second and third experiment were 100, 140, 180 and 200 μg/mL and 1.0, 10, 20, 50, 80, 100, 140 and 200 μg/mL, respectively.
In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 μL.
Care was taken to avoid evaporation of volatile test chemicals and crosscontamination between wells by test chemicals by covering the plates with a foil prior to incubation with the test chemicals
- Precipitate evaluation: After 45 ± 3 hours of exposure, wells were checked for precipitate.
- Cell antibodies staining for IG1 and CD86: Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 μL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 μL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies were used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control;
- Human CD86 specific mouse IgG1.
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 μL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated (4°C) in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and resuspended in 90 μL of PBS.
- Flow cytometry method:
Acquisition:Just before acquisition, 5 μL of a 0.5 μg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
Analysis: All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the propidium iodide (PI) negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity was analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed so that at least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
Colour interferences:There is colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).

ACCEPTABILITY CRITERIA
The U-SENS™ test is considered acceptable if it meets the following criteria:
- At the end of the incubation treatment period, the mean viability of the triplicate untreated (RPMI) U937 cells is > 90%
- No drift in CD86 expression is observed. A drift is defined by
i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50% of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and
ii) the corrected %CD86+ value of the negative control replicate 3 is less than 50% of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
- The CD86 basal expression of untreated (RPMI) U937 cells is within the range of ≥ 2% and ≤ 25%.
- When DMSO is used as a vehicle, 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 is > 90%. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
- The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
- Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70%).

DATA EVALUATION AND STATISTICAL PROCEDURES
- CV70 calculations: For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well.
The theoretical concentration at which the chemical induces 30% cytotoxicity (i.e., 70% viability) was calculated using the following formula:

CV70 = C1+[(V1 – 70)/ (V1 – V2) x (C2 – C1)]

Where:
V1 = the first percentage of viability above 70%
V2 = the first percentage of viability below 70%
C1 = dose level corresponding to V1
C2 = dose level corresponding to V2

- EC150 calculations:
For each CD86 well culture, the percentage of induced CD86+ cells was calculated as:
[absolute %CD86+ — absolute%IgG1+]

A stimulation index (S.I.) is calculated for each dose level as follows:

S.I. = [%CD86+ - %IgG1+] in the treated culture/ Mean [%CD86+ - %IgG1+] of the vehicle cultures x 100

The viability for each dose level are the mean of the IgG1 well and CD86 well. The theoretical concentration at which the chemical induces a S.I. of 150 (i.e., 50% of CD86+ cells over the vehicle control) was calculated using the following formula:

EC150 = C1 + (150 – S.I. 1)/(S.I. 2 – S.I. 1) x (C2 – C1)

Where:
S.I.1 = the first percentage of CD86+ below 150%
S.I.2 = the first percentage of CD86+ above 150%
C1 = dose level corresponding to S.I. 1
C2 = dose level corresponding to S.I. 2

DATA INTERPRETATION
For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
- The individual conclusion of a U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150% at all non-cytotoxic concentrations (cell viability ≥ 70%) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dotplot (IgG1 FL1 Geo Mean S.I. ≥ 150%).
- In all other cases: S.I. of CD86 higher or equal to 150% and/or interferences observed, the individual conclusion of a U-SENS™ run is considered Positive (hereinafter referred to as P).
- A U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative. If the first two runs are both negative (N), the USENS ™ prediction is considered NEGATIVE and a third run does not need to be conducted.
- A U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive. If the first two runs are both positive (P), the USENS™ prediction is considered POSITIVE and a third run does not need to be conducted.
- There is an exception if, in the first run, the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION, and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150%, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4).

Positive control results:

Experiment 1
The positive control (TNBS) showed a S.I. ≥ 200% in all wells and was noncytotoxic at all concentrations (cell viability ≥ 70%). One positive control well showed a S.I. below the positive historical control data.

Experiment 2
The positive control (TNBS) showed a S.I. ≥ 574% in all wells and was noncytotoxic at all concentrations (cell viability ≥ 70%).

Experiment 3
The positive control (TNBS) showed a S.I. ≥ 521% in all wells and was noncytotoxic at all concentrations (cell viability ≥ 70%). One positive control well showed a S.I. above the positive historical control data.

Run / experiment:

other: Experiment 1

Parameter:

other: CV70 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 1

Parameter:

other: EC150 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: CV70 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: EC150 (µg/mL)

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: CV70 (µg/mL)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: EC150 (µg/mL)

Value:

96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The U-SensTM assay was successfully implemented and validated, and lab proficiency has been shown by obtaining the expected U-SensTM prediction for the 10 proficiency chemicals that are described in the OECD 442E guideline.

ACCEPTANCE OF RESULTS:
All tests passed the acceptance criteria:
- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (99% in experiment 1 and 2 and 100% in experiment 3).
- No drift in CD86 expression was observed in the untreated controls and negative controls.
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in the three experiments.
- The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (99% in experiment 1 and 100% in experiment 2 and 3).
- The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in the three experiments.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in the three experiments.

In all experiments the positive, negative and vehicle control were considered valid and within the historical control data, except for one positive control well in experiment 3 which was above the historical control data. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Test item results:

Experiment 1

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

- JNJ-39005525-AAA (EMME) showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be

calculated and was considered to be higher than 200 μg/mL.

- No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with JNJ-39005525-

AAA (EMME). No EC150 could be calculated and was considered to be higher than 200 μg/mL.

- The test item showed no colour interference.

- The negative control (LA) showed a S.I. ≤ 109% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

JNJ-39005525-AAA (EMME) is classified negative as it showed no toxicity (no CV70 value) and no biologically relevant induction of the CD86 activity (no EC150 value) was measured at any of the test concentrations.

Experiment 2

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

- JNJ-39005525-AAA (EMME) showed no toxicity, therefore no CV70 values could be calculated and was considered to be higher than 200 μg/mL

- A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 was < 100 μg/mL.

- The test item showed no colour interference.

- The negative control (LA) showed a S.I. ≤ 96% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

JNJ-39005525-AAA (EMME) is classified as positive in this experiment since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

Experiment 3

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

- JNJ-39005525-AAA (EMME) showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and was considered to be higher than 200 μg/mL.

- A biologically relevant increase in the expression of CD86 was observed after

treatment with the test item, the EC150 was 96 μg/mL.

- The test item showed no colour interference.

- The negative control (LA) showed a S.I. ≤ 121% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

JNJ-39005525-AAA (EMME) is classified as positive in this experiment since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control.

Historical Control Data for the U-SENSTM Assay

	Positive control		Negative control		Vehicle control (RPMI)		Vehicle control (DMSO)
	S.I. (%)	Viability (%)	S.I. (%)	Viability (%)	S.I. (%)	Viability (%)	S.I. (%)	Viability (%)
Range	119 – 749	89 – 103	54 – 132	95 – 101	71 – 129	95 – 102	74 – 227	96 – 102
Mean	434	96	93	98	100	98	151	99
SD	158	3.5	20	1.6	15	1.8	38	1.5
n	240	240	239	239	237	237	174	174

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Nov 2017 to Jul 2018.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

JNJ-39005525-AAA (EMME) is classified as positive in the U-Sens™ assay since positive results (> 150% increase) were observed in 2 out of 3 experiments at test concentrations with a cell viability of >70% compared to the vehicle control.
In conclusion, JNJ-39005525-AAA (EMME) is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Reference 5

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

8 September 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

1. SOFTWARE
Derek Nexus

2. MODEL (incl. version number)
Derek Nexus: 5.0.2, Nexus: 2.1.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCOC=C(C(=O)OCC)C(=O)OCC

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
see attached QMRF
Q13-46-0045
The corresponding QMRF named “Derek for Windows - Skin sensitization” has been downloaded from the JRC QSAR model Database.

5. APPLICABILITY DOMAIN (see Derek report in field 'Attached background material')
Alert matched: 425 Enol ether
Alert matched: 481 alpha,beta-Unsaturated ester or precursor
Skin sensitisation in mammal is PLAUSIBLE

6. ADEQUACY OF THE RESULT (see Derek report in field 'Attached background material')
- Alert 425 Enol ether
Predicted value: The predicted EC3 value of 21% corresponds to “weak sensitizer”
Following EU CLP criteria, if measured experimentally, the predicted value would correspond to “Category 1B” in the CLP classification system as the predicted EC3 value is higher than 2%.

- Alert 481 alpha,beta-Unsaturated ester or precursor
Predicted value: The predicted EC3 value of 0.58% corresponds to “strong sensitizer”
Following EU CLP criteria, if measured experimentally, the predicted value would correspond to “Category 1A” in the CLP classification system as the predicted EC3 value is below 2%.

Principles of method if other than guideline:

- Software tool(s) used including version: Derek Nexus: 5.0.2; Nexus: 2.1.1
- Model(s) used: Knowledge Base: Derek KB 2015 2.0
- Model description: see QMRF in field 'Attached justification'
- Justification of QSAR prediction: the QSAR prediction for skin sensitisation is used as part of the weight-of-evidence approach to cover the information requirements for this endpoint. The justification is further elaborated in the weight-of-evidence justification attached to the skin sensitisation endpoint summary in this dossier.

Specific details on test material used for the study:

SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: CCOC=C(C(=O)OCC)C(=O)OCC
- Average Mol Mass: 216.23
- Exact Mol Mass: 216.0998
- Log Kp: -2.59
- Log P: 2.04
- Composition / purity: other information: not applicable for in silico study

Parameter:

EC3

Value:

21

Test group / Remarks:

Predicted EC3 value (Derek EC3 Model 1.0.6) - alert matched: 425 Enol ether

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Parameter:

EC3

Value:

0.58

Test group / Remarks:

Redicted EC3 value (Derek EC3 Model 1.0.6) - alert matched: 481 alpha,beta-Unsaturated ester or precursor

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Reasoning summary:

Skin sensitisation in mammal is PLAUSIBLE

Alert matched: 425 Enol ether

Alert matched: 481 alpha,beta-Unsaturated ester or precursor

Comments:

Potential mechanism (enol ether): Pre-/pro-hapten producing electrophile with  Schiff base forming potential [Aptula and Roberts]

Potential mechanism (alpha,beta-unsaturated ester): Hapten acting as an electrophilic Michael acceptor [Aptula and Roberts]

References (enol ether):

 Aptula AO and Roberts DW. (2006)

Mechanistic applicability domains for nonanimal-based prediction of toxicological end points: general principles and application to reactive toxicity., Chemical Research in Toxicology, 19 , 1097-1105

DOI: 10.1021/tx0601004  

  European Chemicals Agency (ECHA). (1991)

Skin sensitisation study for 1,4-bis[(vinyloxy)methyl]cyclohexane (CAS No. 17351-75-6)., European Chemicals Agency Registration Dossier,  ,

References (alpha,beta-unsaturated ester or precursor):

  Basketter DA and Scholes EW. (1992)

Comparison of the local lymph-node assay with the guinea-pig maximization test for the detection of a range of contact allergens., Food and Chemical Toxicology, 30 , 65-69

DOI: 10.1016/0278-6915(92)90138-B  

  Cronin MTD and Basketter DA. (1994)

Multivariate QSAR analysis of a skin sensitization database., SAR and QSAR in Environmental Research, 2 , 159-179

DOI: 10.1080/10629369408029901  

  Hostynek JJ, Lauerma AI, Magee PS, Bloom E and Maibach HI. (1995)

A local lymph-node assay validation study of a structure-activity relationship model for contact allergens., Archives of Dermatological Research, 287 , 567-571

DOI: 10.1007/BF00374078  

  Barbier P and Benezra C. (1986)

Allergenic alpha-methylene-gamma-butyrolactones.  Study of the capacity of beta-acetoxy- and beta-hydroxy-alpha-methylene-gamma-butyrolactones to induce allergic contact dermatitis in guinea pigs., Journal of Medicinal Chemistry, 29 , 868-871

  Turk JL, Parkers D, Long PV and Bull JE. (1986)

Induction of immunologic tolerance: desensitisation to occupational allergens., Journal of Allergy and Clinical Immunology, 78 , 1082-1085

  Roberts DW. (1987)

Structure-activity relationships for skin sensitisation potential of diacrylates and dimethacrylates., Contact Dermatitis, 17 , 281-289

  Ashby J, Basketter DA, Paton D and Kimber I. (1995)

Structure activity relationships in skin sensitization using the murine local lymph node assay., Toxicology, 103 , 177-194

DOI: 10.1016/0300-483X(95)03132-Y  

  Kanazawa Y, Yoshida T and Kojima K. (1999)

Structure-activity relationships in allergic contact dermatitis induced by methacrylates: studies of the influence of side-chain length of methacrylates., Contact Dermatitis, 40 , 19-23

  Franot C, Roberts DW, Smith RG, Basketter DA, Benezra C and Lepoittevin JP. (1994)

Structure-activity relationships for contact allergenic potential of gamma,gamma-dimethyl-gamma-butyrolactone derivatives.  1.  Synthesis and electrophilic reactivity studies of alpha-(omega-substituted-alkyl)-gamma,gamma-dimethyl-gamma-butyrolactones ..., Chemical Research in Toxicology, 7 , 297-306

  Franot C, Roberts DW, Basketter DA, Benezra C and Lepoittevin JP. (1994)

Structure-activity relationships for contact allergenic potential of gamma,gamma-dimethyl-gamma-butyrolactone derivatives.  2.  Quantitative structure-skin sensitization relationships for ..., Chemical Research in Toxicology, 7 , 307-312

  Opdyke DLJ. (1976)

Monographs on fragrance raw materials: diethyl maleate., Food and Cosmetics Toxicology, 14 , 443-444

  Aptula AO and Roberts DW. (2006)

Mechanistic applicability domains for nonanimal-based prediction of toxicological end points: general principles and application to reactive toxicity., Chemical Research in Toxicology, 19 , 1097-1105

DOI: 10.1021/tx0601004  

Validation comments (enol ether):

Skin sensitisation: guinea pig maximisation test, local lymph node assay

The alert has demonstrated the following predictive performance:

1) Cronin and Basketter: 0 compounds activate this alert.

2) Gerberick: 0 compounds activate this alert.

3) Contact Dermatitis: 0 compounds activate this alert.

1) A collection of guinea pig maximisation test data for 216 compounds from the following reference: Cronin MTD and Basketter DA. Multivariate QSAR analysis of a skin sensitization database. SAR and QSAR in Environmental Research, 1994, 2, 159-179, available at "http://dx.doi.org/10.1080/10629369408029901".

2) A collection of local lymph node assay data for 318 compounds derived from the following references: (i) Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, Patlewicz GY and Basketter DA. Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis, 2005, 16, 157-202. Downloaded from "http://www.inchemicotox.org/results/" (3 September 2010); (ii) Kern PS, Gerberick GF, Ryan CA,

Kimber I, Aptula A and Basketter DA. Local lymph node data for the evaluation of skin sensitization alternatives: a second compilation. Dermatitis, 2010, 21, 8-32, available at &ldquo;http://dx.doi.org/10.2310/6620.2009.09038&rdquo;.

3) A collection of local lymph node assay data for 137 compounds published in Contact Dermatitis which have been extracted from Vitic Nexus (13 September 2012).

Validation comments (alpha,beta-unsaturated ester or precursor):

Skin sensitisation: guinea pig maximisation test, local lymph node assay

The alert has demonstrated the following predictive performance:

1) Cronin and Basketter: 3 compounds activate this alert of which 2 are reported positive. (Positive predictivity: 67%.)

2) Gerberick: 9 compounds activate this alert of which 6 are reported positive. (Positive predictivity: 67%.)

3) Contact Dermatitis: 8 compounds activate this alert of which 3 are reported positive. (Positive predictivity: 38%.)

1) A collection of guinea pig maximisation test data for 216 compounds from the following reference: Cronin MTD and Basketter DA. Multivariate QSAR analysis of a skin sensitization database. SAR and QSAR in Environmental Research, 1994, 2, 159-179, available at "http://dx.doi.org/10.1080/10629369408029901".

2) A collection of local lymph node assay data for 318 compounds derived from the following references: (i) Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, Patlewicz GY and Basketter DA. Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods. Dermatitis, 2005, 16, 157-202. Downloaded from "http://www.inchemicotox.org/results/" (3 September 2010); (ii) Kern PS, Gerberick GF, Ryan CA,

Kimber I, Aptula A and Basketter DA. Local lymph node data for the evaluation of skin sensitization alternatives: a second compilation. Dermatitis, 2010, 21, 8-32, available at &ldquo;http://dx.doi.org/10.2310/6620.2009.09038&rdquo;.

3) A collection of local lymph node assay data for 137 compounds published in Contact Dermatitis which have been extracted from Vitic Nexus (13 September 2012).

EC3 Result for Derek EC3 Model - 1.0.6:

- Alert matched: 425 Enol ether

Predicted LLNA EC3: 21% (weak sensitiser)

Experimental Match: No exact match found

Compounds used in calculation: 3/3

- Alert matched 481: alpha,beta-Unsaturated ester or precursor

Predicted LLNA EC3: 0.58% (strong sensitiser)

Experimental Match: No exact match found

Compounds used in calculation: 10/25

Interpretation of results:

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

Conclusions:

EMME is predicted as a sensitiser to the skin with a predicted LLNA EC3 value of 0.58 (alpha,beta-unsaturated ester or precursor) and 21% (enol ether) and a prediction strength as Plausible using Derek Nexus v5.0.2. Based on the worst case predicted EC3 value of 0.58%, EMME is classified as skin sensitiser category 1A. This substance triggered a skin sensitisation alert for 425 enol ether and 481 alpha,beta-unsaturated ester or precursor.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In silico, in chemico and in vitro data are used in a weight-of-evidence approach. The studies are discussed in detail in the Weight-of-Evidence justification attached to this endpoint summary.

 

In addition, the Local Lymph Node Assay (LLNA) is added as key study (van Sas, 2018).

 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 0.1, 0.2 or 0.5% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

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.

All auricular lymph nodes of the animals of the control group, two animals treated at 0.2% and all animals treated at 0.5% were considered normal in size. The auricular lymph nodes of all animals treated at 0.1% and three animals treated at 0.2% were considered to be enlarged, which is in line with the increased DPM values for these groups. 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 0.1, 0.2 and 0.5% were 1633, 2352 and 3242 DPM, respectively. The mean DPM/animal value for the vehicle control group was 651 DPM. The SI values calculated for the item concentrations 0.1, 0.2 and 0.5% were 2.5, 3.6 and 5.0, respectively.

 

These results show that the test item elicits a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 0.15% was calculated.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Taking into account the positive in silico, in chemico results and the positive results obtained in a second in vitro assay and the fact that it was not possible to conclude on the skin sensitizing properties (or potency) of the test item based on non-animal testing approaches, as required in Annex VII, section 8.3, additional in vivo testing (Local Lymph Node Assay in mice according to OECD 429) was performed to assess the potency of the test item.

Based on the results of the Local Lymph Node Assay in female CBA mice:

- according to the recommendations made in the test guidelines (including all amendments), JNJ-39005525-AAA (EMME) would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), JNJ-39005525-AAA (EMME) should be classified as skin sensitizer (Category 1A).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), JNJ-39005525-AAA (EMME) should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.