Registration Dossier

Administrative data

Description of key information

Skin sensitisation:

-In vivo:a Local Lymph Node Assay (LLNA) was performed and the test item was found to be skin sensitising and classified as skin sensitizer (Category 1A).

In silico and in chemico data are used in weight-of-evidence approach.

- In silico: A Derek Nexus assessment yielded an alert for skin sensitisation based on the presence of a sulphonyl halide (Alert 403) (De Vlieger, 2017). No EC3 prediction based on data of nearest neighbours was provided for this test item as fewer than three EC3 data points were available.

- In chemico: The test item was considered to be positive in the DPRA and was classified in the "high reactivity class" when using the Cysteine 1:10 / Lysine 1:50 prediction model.

No KeratinoSensTM assay was performed with this test item as it was found unstable in DMSO and other solvents that could be used in this test (i.e. water, medium and ethanol).

Performance of an additional in vitro assay addressing the activation of dendritic cells (Key event 3) would not yield additional relevant information, irrespective if the study would give a negative or positive result. In addition, the solvents used in this Key event 3 assay are the same those used in the KeratinoSensTM assay in which the test item was observed to be unstable.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vitro
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-09-13 to 2017-09-28
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: M16LB4523
- Expiration date of the lot/batch: 2017-12-01 (retest date)
- Purity (GC): 99.8%

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). In addition, stability of the test item in ACN was confirmed in solvent stock solutions from CRL projects 516401 and 520060. A report of Stability testing in different vehicles for this test item is attached to this entry.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the cysteine and lysine reactivity assay 34.83 mg of the test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1572 µL 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_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 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 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 mean 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.
Parameter:
other: % SPCC depletion
Run / experiment:
Run 1 / mean of 3 replicates
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: % SPCL depletion
Run / experiment:
Run 1/ mean of 3 replicates
Value:
48.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: % mean of SPCC and SPCL depletion
Run / experiment:
Run 1/ mean of 3 replicates
Value:
74.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Positive: high 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: 0.993 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 mM ± 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. This 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% to 100%
- 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. In addition, stability of the test item in ACN was confirmed in solvent stock solutions from CRL projects 516401 and 520060.

Results cysteine reactivity assay for the test item:

Preparation of a 100 mM test item 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, no mean SPCC A220/A258 area ratio could be determined since the test item displayed 100% reactivity towards SPCC. 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 test item stock solution in ACN 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. 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 15.94. Tthis was outside the 12.50 -15.27 range (mean A220/A258 ratio ± 10% range of Reference controls A, B and C). However, since the test item displayed high reactivity towards SPCL, accurate calculation of the peak purity was not possible due to the low SPCL signal at 258 nm. Overall, it can be concluded that the test item did not co-elute 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

 

 100%

 ± 0.0%

 48.4%

 ±2.3%

74.2%

 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.
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
C1(=CC=C(C=C1)[N+]([O-])=O)S(=O)(=O)Cl

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: 403 Sulphonyl halide
Skin sensitisation in mammal is PLAUSIBLE

6. ADEQUACY OF THE RESULT (see Derek report in field 'Attached background material')
No EC3 prediction based on data of nearest neighbours was provided for this test item as fewer than three EC3 data points were available. The prediction showed one similar compound with a similarity of 34%.
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: C1(=CC=C(C=C1)[N+]([O-])=O)S(=O)(=O)Cl
- Average Mol Mass: 221.62
- Exact Mol Mass: 220.955
- Log Kp: -4.14
- Log P: -0.09
- Composition / purity: other information: not applicable for in silico study
Parameter:
other: no prediction on EC3
Test group / Remarks:
No EC3 prediction based on data of nearest neighbours was provided for this test item as fewer than three EC3 data points were available. The prediction showed one similar compound with a similarity of 34%.
Remarks on result:
positive indication of skin sensitisation based on QSAR/QSPR prediction

Reasoning summary:

Skin sensitisation in mammal is PLAUSIBLE

Alert matched: 403 Sulphonyl halide

Comments:

Potential mechanism: Hapten acting as an electrophilic sulphonylating agent [Payne and Walsh]

References:

  Landsteiner K and Jacobs J. (1936)

Studies on the sensitization of animals with simple chemical compounds.  II., Journal of Experimental Medicine, 64 , 625-639

DOI: 10.1016/S0021-8707(36)90150-8  

  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  

  Payne MP and Walsh PT. (1994)

Structure-activity relationships for skin sensitization potential: development of structural alerts for use in knowledge-based toxicity prediction systems., Journal of Chemical Information and Computer Sciences, 34 , 154-161

DOI: 10.1021/ci00017a019  

Validation comments:

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

The alert has demonstrated the following predictive performance:

1) Cronin and Basketter: 1 compound activates this alert of which 1 is reported positive. (Positive predictivity: 100%.)

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).

EC3 Result for Derek EC3 Model - 1.0.5:

Predicted LLNA EC3: No EC3 prediction based on data of nearest neighbours was provided for this test item as fewer than three EC3 data points were available. The prediction showed one similar compound with a similarity of 34%.

Experimental Match: No exact match found

Compounds used in calculation: 1/1

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
No EC3 prediction based on data of nearest neighbours was provided for this test item as fewer than three EC3 data points were available. Skin sensitisation is Plausible using Derek Nexus v 5.0.2. Therefore, 4-nitrobenzene-1-sulfonyl chloride is classified as skin sensitiser category 1. This substance triggered a skin sensitisation alert for 403 Sulphonyl halide.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-14 to 2018-03-219
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Paris Cedex, July 2010
Deviations:
no
Qualifier:
according to
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:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
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: M16LB4523
- Expiration date of the lot/batch: 2018-06-23 (retest date)
- Purity/composition correction factor: 1
- Purity: 99.9% w/w (calculated as is) - Chromatographic purity 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: There was no information available regarding the solubility or stability in vehicle.

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:
inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/J strain, inbred, SPF-Quality
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Approximately 8 weeks old
- Weight at study initiation: 17.8 - 22.7 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 of the main study, 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.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 18 to 24°C; actual daily mean temperature during the study period: 22°C
- Humidity (%): 40 to 70%; actual main relative humidity during the study period: 40 to 45%
- Air changes (per hr): At least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark
Vehicle:
methyl ethyl ketone
Concentration:
PreScreen Test: 25 and 50% w/w
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (5% and 10%) at a later stage.

Main Study: 0, 1, 2 and 5% w/w
No. of animals per dose:
Five females per group; 4 groups (including control group)
Details on study design:
Rationale vehicle selection:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methyl ethyl ketone, propylene glycol and dimethylsulfoxide. Formulation in Methyl ethyl ketone has proven to be stable in a separate study.

PRE-SCREEN TESTS:
- At a 10, 25 and 50% test item concentration variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values and therefore these concentrations did not meet the selection criteria.
- Compound solubility: no data
- Irritation: At a 5% test item concentration no to very slight irritation was observed
- Systemic toxicity: At a 5% test item concentration no signs of systemic toxicity were noted
- Ear thickness measurements: At a 5% test item concentration variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test item concentration selected for the main study was a 5% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
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 (2015) (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 skinreaction
EC3 value ≤ 2%: sub-category 1A
EC3 value > 2%: sub-category 1B
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data
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:
12.6
Variability:
+/- 2.1
Test group / Remarks:
Based on 5 animals in 1% w/w in methyl ethyl ketone group
Parameter:
SI
Value:
18.1
Variability:
+/- 1.6
Test group / Remarks:
Based on 5 animals in 2% w/w in methyl ethyl ketone group
Parameter:
SI
Value:
20.2
Variability:
+/- 1.0
Test group / Remarks:
Based on 5 animals in 5% w/w in methyl ethyl ketone group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 821 ± 118
1% w/w group: mean DPM ± SEM: 10345 ± 1743
2% w/w group: mean DPM ± SEM: 14891 ± 1311
5 %w/w group: mean DPM ± SEM: 16562 ± 817
SEM = Standard Error of the Mean

EC3 CALCULATION
The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 0 and 1%.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: The scaliness as shown by one animal treated at a concentration of 2% and all animals treated at a concentration of 5% on days 4 and 6 were considered not to have a toxicologically significant effect on the activity of the nodes.
- 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 groups were considered enlarged compared to the control group. The largest auricular lymph nodes were found in the higher dose groups. 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 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
These results show that the test item elicits a SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 0 and 1%.
Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), 4-Nitrobenzene-1-sulfonyl chloride 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), 4-Nitrobenzene-1-sulfonyl chloride 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), 4-Nitrobenzene-1-sulfonyl chloride should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A Local Lymph Node Assay (LLNA) has been performed and based on the EC3 value calculated, the test item should be classified as skin sensitizer (Category 1A).

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 1, 2 or 5% w/w in methyl ethyl ketone on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).

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 experimental groups were considered enlarged compared to the control group. The largest auricular lymph nodes were found in the higher dose groups No macroscopic abnormalities of the surrounding area were noted for any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals during the study.

Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 10345, 14891 and 16562 DPM, respectively. The mean DPM/animal value for the vehicle control group was 821 DPM. The SI values calculated for the item concentrations 1, 2 and 5% were 12.6, 18.1 and 20.2, respectively.

These results show that the test item elicits a SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 0 and 1%. Based on the results, 4-Nitrobenzene-1-sulfonyl chloride is classified as a skin sensitizer Category 1A according to CLP.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In conclusion, taking into account (1) the positive in silico and in chemico 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 (LLNA) in mice according to OECD 429) has been performed to assess the potency of the test item. Based on the EC3 value calculated in the LLNA, according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015)(including all amendments) and to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), 4-Nitrobenzene-1-sulfonyl chloride should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.