Registration Dossier

Administrative data

Description of key information

Skin sensitisation:

In silico, in chemico and in vitro data are used in a weight-of-evidence approach. 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 an Alkyl halide group (Alert 413) and predicted an EC3 of 0.075% (Van Gompel, 2017).

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

- In vitro (OECD 442D): The KeratinoSensTM assay yielded a positive result (Westerink, 2018).

Taking into account the positive in silico, in chemico and in vitro results and as 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) was performed to assess the potency of the test item (van Sas, 2018).

- In vivo (OECD 429): Based on the results of a local Lymph Node Assay in female CBI/J mice the test item was regarded as a skin sensitizer category 1B

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-12-06 to 2018-01-29
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16DB2043
- Expiration date of the lot/batch: 25 April 2020 (retest date)
- Purity: 99% (base titration - assay)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: No data. Homogeneity was assessed by visual inspection of the formulations

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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 was not applicable, since the test method required a logical concentration range rather than specific dose levels to be dosed.

Species:
mouse
Strain:
CBA:J
Remarks:
CBA/J strain, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: young adults (approx. 10 weeks old)
- Weight at study initiation: 19.6 - 23.7 grams
- Housing: Group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS- J. Rettenmaier & Söhne GmbH + Co. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd., USA) 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 Spezialdiaten 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C; actual daily mean: 22 to 23 °C
- Humidity (%): 40 to 70%; actual daily mean: 42 to 46%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
propylene glycol
Concentration:
pre-screen test: 25 and 50% w/w (10 and 25% w/w at later stage based on the results of the initially treated animals)
main study: 0, 2, 10 and 25% w/w
No. of animals per dose:
Pre-screen test: 2 females per group, 4 groups (1 excluded from results)
Main study: 5 females per group; 4 groups
Details on study design:
Rationale vehicle:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v) (test item did not dissolve), N,N-dimethylformamide (test item did not dissolve), methylethylketone (test item did not dissolve), propylene glycol (thin paste/turbid solution) and dimethylsulfoxide (turbid solution). Propylene glycol appeared visually as the most homogenous formulation and was therefore used as vehicle in this study.

PRE-SCREEN TESTS:
- Systemic toxicity: At a 25% and 10% test item concentration no signs of toxicity or excessive irritation were noted.
- Ear thickness measurements: At a 50% test item concentration, variation in ear thickness did not meet the selection criteria. At a 25% test item concentration, variation in ear thickness was within
the selection criteria. At a 10% test item concentration, variation in ear thickness for one animal exceeded the selection criteria while the other animal did not exceed the selection criteria. Since only one animal at 10% and no animals at 25% did not meet the selection criteria, 25% was selected as the highest concentration to be used in the main study.
- Erythema scores: 0 = no erythema in all animals

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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 (2011) (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. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3).
Classification of results:
SI value UN-GHS 2015; 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

TREATMENT PREPARATION AND ADMINISTRATION:
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistics performed
Positive control results:
At concentrations 5%, 10% and 25% SI values of the positive control item were 1.4, 2.2, and 3.5 respectively. An EC3 value of 19.2% was calculated using linear interpolation. The calculated EC3 value was in the accepable range of 4.8 and 19.5%. The results of the 6 monthly reliability checks of the recent years were 13.2, 14.1, 17.3, 9.8, 17.8, 18.0, 14.7 and 13.2%
Based on the results, it was concluded that the Local Lymph Node Assay as performed in the laboratory is an appropriate model for testing contact hypersensitivity.
Parameter:
SI
Value:
1.5
Variability:
+/- 0.4
Test group / Remarks:
Based on 5 animals in 2% w/w in propylene glycol group
Parameter:
SI
Value:
1.7
Variability:
+/- 0.4
Test group / Remarks:
Based on 5 animals in 10% w/w in propylene glycol group
Parameter:
SI
Value:
3.6
Variability:
+/- 1.3
Test group / Remarks:
Based on 5 animals in 25% w/w in propylene glycol group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
0% w/w group: mean DPM/animal: 610 ± 46
2% w/w group: mean DPM/animal: 937 ± 237
10% w/w group: mean DPM/animal: 1026 ± 240
25%w/w group: mean DPM/animal: 2205 ± 792

DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the item concentrations 2, 10 and 25% were 1.5, 1.7 and 3.6, respectively.

EC3 CALCULATION
These results indicate that the test item could elicit 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 20.3% was calculated.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation:
No irritation of the ears was observed in any of the animals examined.
White test item remnants were present on the dorsal surface of the ears of all animals treated at 10% and 25% between Days 1 and 3, 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 of the main study.
- Macroscopy of the auricular lymph nodes and surrounding area:
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. 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.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
These results indicate that the test item could elicit 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 20.3% was calculated.

Based on these results:
-According to the recommendations made in the test guidelines (including all amendments), T001250 would be regarded as skin sensitizer.
-According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), T001250 should be classified as skin sensitizer (Category 1B).
- According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), T001250 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-05-23 to 2017-06-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16DB2043
- Expiration date of the lot/batch: 2020-04-25 (retest date)
- Purity (GC): 98.4%

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 solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ) and MQ/ACN (1:1, v/v).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the cysteine and lysine reactivity assay 32.66 mg of the test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1467 µL MQ/ACN (1:1, v/v) 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 was made for the purity/composition of the test item as the correction factor is 1.00.
Details on 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_MHeW0217
- Batch SPCL: 220114HSDW_W0217
- 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 20 mg of SPCC in 39.92 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. In addition, a RCcysCwater/ACN sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCwater/ACN sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL MQ/ACN (1:1, v/v).
- 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 20 mg of SPCL in 38.61 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. In addition, a RClysCwater/ACN sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCwater/ACN sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL MQ/ACN (1:1, v/v).
- 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 25 and 26 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 13 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature.

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 Replication 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.
There might be cases where the test item absorbed significantly at 220 nm and had the same retention time as the peptide (co-elution). If co-elution of a test item occurred with both the cysteine and the lysine peptide then the analysis was reported as “inconclusive”. In the case where co-elution occurred only with the lysine peptide, the Cysteine 1:10 prediction model was used.
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 73.1% ± 1.4%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). This was also within the historical positive control data range.

Lysine reactivity assay:
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde, calculated versus the mean SPCL peak area of Reference Controls C, was 58.7% ± 4.3%. 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 also within the historical positive control data range.
Parameter:
other: % SPCC depletion
Run / experiment:
Run 1 / mean of 3 replicates
Value:
47.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: positive: moderate reactivity
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: 0.994 was within the acceptance criteria (r²>0.99) (SPCC standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.515 ± 0.012 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.474 mM 0.014 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control Cwater/ACN samples: 0.450 ± 0.022 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A, C and Cwater/ACN were all within the acceptance criteria which confirms the suitability of the HPLC system and indicates that the solvent (MQ/ACN (1:1, v/v)) used to dissolve the test item did not impact the Percent SPCC Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 3.9% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 73.1% was within the acceptance range of 60.8% to100%
- SD of peptide depletion cinnamic aldehyde: 1.4% was below the maximum (SD <14.9%)

ACCEPTANCE OF RESULTS - Lysine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.991 was within the acceptance criteria (r²>0.99) (SPCL standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.501 ± 0.006 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.484 ± 0.006 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control Cwater/ACN samples: 0.480 ± 0.011 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A, C and Cisopropanol were all within the acceptance criteria which confirms the suitability of the HPLC system and indicates that the solvent (MQ/ACN (1:1, v/v)) used to dissolve the test item did not impact the Percent SPCL Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 2.6% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 58.7% was within the acceptance range of 40.2% to 69.0%
- SD of peptide depletion cinnamic aldehyde: 4.3% was below the maximum (SD <11.6%)

Solubility assessment:

At a concentration of 100 mM, the test item was not soluble (precipitation) in ACN and MQ, but was soluble in MQ/ACN (1:1, v/v). Therefore this solvent was used to dissolve the test item in this DPRA study.

Results cysteine reactivity assay for the test item:

Preparation of a 100 mM test item stock solution in MQ/ACN (1:1, v/v) 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 19.04 which was within the 15.77 -19.27 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 in SPCC.

Results lysine reactivity assay for the test item:

Preparation of a 100 mM test item stock solution in MQ/ACN (1:1, v/v) showed that the test item dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a precipitate was observed in the CC and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide. Consequently, the peptide depletion might be underestimated.

In the CC sample a peak/interference was observed at the retention time of SPCL. For the test item samples, the mean SPCL A220/A258 area ratio was 8.28. Since this was outside the 12.22 -14.93 range (mean A220/A258 ratio ± 10% range of Reference controls A, B and C), this

again indicated that there was co-elution of the test item with SPCL and therefore no SPCL Depletion value could be calculated.

SPCC and SPCL depletion, DPRA prediction and reactivity classification of the test item:

    SPCC depletion     SPCL depletion     DPRA prediction and reactivity classification
 Mean  ± SD  Mean  ± SD  Cysteine 1:10
 47.4%  ± 5.8% NA: Co-elution    

 Positive:

moderate reactivity

SD = Standard Deviation; NA = not applicable              

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 “moderate reactivity class” when using the Cysteine 1:10 prediction model.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-08-09 to 2017-08-25
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: I16DB2043
- Expiration date of the lot/batch: 2020-04-25 (retest date)
- Purity (base titration): 99%

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 (clear yellow solution). The stock was sonicated for 31 minutes with a temperature of 19.0 – 37.0 ºC to dissolve the test item. The 100-fold dilution of the 200 mM DMSO stock in exposure medium also formed a clear solution (final concentration 2000 μM). 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: IIn the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. The stock was sonicated for 10 minutes at 29.0 – 34.0 ºC in experiment 1 and for 1 minute at 35.0 ºC in experiment 2. From the 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 1.00.
Details on 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. The stock was sonicated for 10 minutes at 29.0 – 34.0 ºC in experiment 1 and for 1 minute at 35.0 ºC in experiment 2. From the 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 1.5 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, 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.

PREPARATION OF THE SOLVENT CONTROL
The solvent control was 1% DMSO in exposure medium. Sixteen wells were tested per plate (2 of the luminescence readings for the solvent control were excluded from the analysis in experiment 2, since the variation was with 21.6% > 20%. Based on evaluation of the data with Minitab 17 (Minitab Inc., Coventry, United Kingdom) these values were clear outliers.

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 81 – 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.9 – 37.1°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 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
- Two experiments were conducted
- 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, 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 24 ± 1 hours. 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 exposure medium) 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 at 37±1.0°C in the presence of 5% CO2. In total 2 experiments were performed.
- 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 DMEM-glutamax containing 5 mg/mL 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 minimum of two experiments was conducted, in case of two not concordant results, a third experiment was performed. 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 (50 μM and 47 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.5-fold and 2.2-fold in experiment 1 and 2, respectively).
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
37.32
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
20.17
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC30 (in µM)
Run / experiment:
Experiment 1
Value:
730
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC30 (in µM)
Run / experiment:
Experiment 2
Value:
992
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: EC1.5
Run / experiment:
Experiment 1
Value:
279
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: EC1.5
Run / experiment:
Experiment 2
Value:
405
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50
Run / experiment:
Experiment 1
Value:
902
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50
Run / experiment:
Experiment 2
Value:
1 388
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
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:
Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control 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 (50 µM and 47 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.5-fold and 2.2-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% (4.6% and 8.8% 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 toxicity. The calculated IC30 was 730 μM and the calculated IC50 was 902 µM.

- A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 37 and the EC1.5 279 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 toxicity. The calculated IC30 was 992 μM and the calculated IC50 was 1388 µM.

- A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 21 and the EC1.5 405 μM.

Historical Control Data for the KeratinoSensTM Studies:

      Positive control
   EC1.5 (µM) Imax (µM) 
Range 5.3 - 123.0  1.6 - 52.7 
Mean  50.9  3.7 
 SD 29.2  4.8 
 n 193  189 

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 December 2017.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, JNJ-559728-AAA (T001250) is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.
Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
25 April 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: 5.0.1, Nexus: 2.1.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC2=C(CCCl)C(=O)N1C=CC=CC1=N2

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
see attached QMRF
Q13-34-36-315
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: 413 Alkyl halide
Skin sensitisation in mammal is PLAUSIBLE

6. ADEQUACY OF THE RESULT (see Derek report in field 'Attached background material')
Predicted value: The predicted EC3 value of 0.075% corresponds to “extreme 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: 5.0.1; Nexus: 2.1.0
- Model(s) used: Knowledge Base: Derek KB 2015 1.0
- 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: CC2=C(CCCl)C(=O)N1C=CC=CC1=N2
- Average Mol Mass: 222.67
- Exact Mol Mass: 222.056
- Log Kp: -3.16
- Log P: 1.29
- Composition / purity: other information: not applicable for in silico study
Parameter:
EC3
Value:
0.075
Test group / Remarks:
Predicted EC3 value (Derek EC3 Model 1.0.5)
Remarks on result:
positive indication of skin sensitisation based on QSAR/QSPR prediction

Reasoning summary:

Skin sensitisation in mammal is PLAUSIBLE

Alert matched: 413 Alkyl halide

Comments:

Potential mechanism: Hapten acting as an SN2 - reactive electrophile [Aptula and Roberts]

References:

- Basketter DA, Roberts DW, Cronin M and Scholes EW. (1992)

The value of the local lymph node assay in quantitative structure-activity investigations., Contact Dermatitis, 27, 137-142

DOI: 10.1111/j.1600-0536.1992.tb05241.x

-  Rao KS, Betso JE and Olson KJ. (1981)

A collection of guinea pig sensitization test results grouped by chemical class., Drug and Chemical Toxicology, 4, 331-351

DOI: 10.3109/01480548109017825

-  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

-  Smith CK and Hotchkiss SAM. (2001)

Xenobiotics as skin sensitisers: metabolic activation and detoxication, and protein-binding mechanisms., Allergic Contact Dermatitis: Chemical and Metabolic Mechanisms, , 119-205

-  Basketter DA, Roberts D, Cronin MTD and Scholes E. (1993)

The value of the local lymph node assay in the development of QSARs for skin sensitization potential., Trends in QSAR and Molecular Modelling 92: 9th European Symposium on Structure-Activity Relationships: QSAR and Molecular Modelling: Papers and Summaries, , 580-583

- European Chemical Agency (ECHA). (2009)

Skin sensitisation study for 1-bromopentane (CAS No. 110-53-2)., European Chemicals Agency Registration Dossier,

European Chemical Agency (ECHA). (2003)

Skin sensitisation study for 1,6-dibromohexane (CAS No. 629-03-8)., European Chemicals Agency Registration Dossier

- Siegel PD, Fedorowicz A, Butterworth L, Law B, Anderson SE, Snyder J and Beezhold D. (2009)

Physical-chemical and solvent considerations in evaluating the influence of carbon chain length on the skin sensitization activity of 1-bromoalkanes., Toxicological Sciences, 107 , 78-84

DOI: 10.1093/toxsci/kfn212

- Natsch A, Ryan CA, Foertsch L, Emter R, Jaworska J, Gerberick F and Kern P. (2013)

A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation., Journal of Applied Toxicology, 33 , 1337-1352

DOI: 10.1002/jat.2868

- Esteve E, Bagot M, Joly P, Souteyrand P, Beylot-Barry M, Vaillant L, Delaunay M, Avril MF, Laroche L, Grange F, Thomine E and Wechsler J. (1999)

A prospective study of cutaneous intolerance to topical mechlorethamine therapy in patients with cutaneous T-cell lymphomas., Archives of Dermatology, 135 , 1349-1353

DOI: 10.1001/archderm.135.11.1349

- 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

·        Baeck M, Chemelle JA, Terreux R, Drieghe J and Goossens A. (2009)

Delayed hypersensitivity to corticosteroids in a series of 315 patients: clinical data and patch test results., Contact Dermatitis, 61 , 163-175

DOI: 10.1111/j.1600-0536.2009.01602.x

-  Ashby J, Hilton J, Dearman RJ, Callander RD and Kimber I. (1993)

Mechanistic relationship among mutagenicity, skin sensitization, and skin carcinogenicity., Environmental Health Perspectives, 101 , 62-67

-  Baer RL, Michaelides P and Prestia AE. (1972)

Failure to induce immune tolerance to nitrogen mustard. Intravenous administration preceding topical use in patients with proriasis., Journal of Investigative Dermatology, 58 , 1-4

DOI: 10.1111/1523-1747.ep13063763

-  Roberts DW, Schultz TW, Wolf EM and Aptula AO. (2010)

Experimental reactivity parameters for toxicity modeling: application to the acute aquatic toxicity of SN2 electrophiles to Tetrahymena pyriformis., Chemical Research in Toxicology, 23 , 228-234

DOI: 10.1021/tx9003648

Validation comments:

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: 19 compounds activate this alert of which 15 are reported positive. (Positive predictivity: 79%.)

3) Contact Dermatitis: 11 compounds activate this alert of which 5 are reported positive. (Positive predictivity: 45%.)

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 - 1.0.5:

Predicted LLNA EC3: 0.075% (extreme sensitiser)

Experimental Match: No exact match found

Compounds used in calculation: 10/36

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
T001250 is predicted as extreme sensitiser to the skin with a predicted LLNA EC3 value of 0.075% and a prediction strength as Plausible using Derek Nexus v5.0.1. Therefore, T001250 is classified as skin sensitiser category 1A. This substance triggered a skin sensitisation alert for 413 Alkyl halide.
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.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Propylene glycol).

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. No irritation of the ears was observed in any of the animals examined. White test item remnants were present on the dorsal surface of the ears of all animals treated at 10% and 25% between Days 1 and 3, which did not hamper scoring of the skin reactions.

No irritation of the ears was observed in any of the animals examined. White test item remnants were present on the dorsal surface of the ears of all

animals treated at 10% and 25% between Days 1 and 3, which did not hamper scoring of the skin reactions. 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.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. 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 2, 10 and 25% were 937, 1026 and 2205 DPM, respectively. The

mean DPM/animal value for the vehicle control group was 610 DPM. The SI values calculated for the item concentrations 2, 10 and 25% were 1.5, 1.7 and 3.6,

respectively.

These results indicate that the test item could elicit 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 20.3% was calculated.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation:

Taking into account the positive in silico, in chemico and in vitro results and as 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) was performed to assess the potency of the test item.

Based on the results of a local Lymph Node Assay in female CBI/J mice the test item was regarded as a skin sensitizer category 1B.