reaction products of 1 mole of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs and 1 mole of cyclohexylamine

Endpoint summary

Description of key information

The skin sensitization potential of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine was assessed according to :

- in vitro skin sensitisation assay (Keratinosens assay),

- in chemico skin sensitization (Direct peptide reactivity assay/DPRA),

- skin sensitization test (local lymph node assay/LLNA)

The overall data indicate that the substance has a weak skin sensitiation potential.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2017 - 10 March 2017

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)

Version / remarks:

February 2015

Deviations:

yes

Remarks:

Except that no solubility test was performed since the test item was already formulated in water. This deviation to the guideline was considered not to have any impact on the integrity of the study once the test item was soluble in a recommended vehicle.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Vehicle: water for injections
Negative control: DMSO; this negative control was applied to cells at a concentration of 1% in culture medium.
Positive control item: Cinnamic Aldehyde (CA). For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of two, to obtain a total of five concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 µM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

Criteria: interpretation of results
Acceptance criteria
Each run was considered valid if the following criteria were met:
- the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between two and eight. If the latter criterion was not fulfilled, the dose response of Cinnamic Aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control,
- the average Coefficient of Variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

Run / experiment:

other: mean (first run)

Parameter:

other: EC1.5

Remarks:

expressed in µM

Value:

4.27

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: mean (second run)

Parameter:

other: EC1.5

Remarks:

expressed in µM

Value:

13.72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: first run

Parameter:

other: Imax

Value:

2.16

Run / experiment:

other: Second run

Parameter:

other: Imax

Value:

3.97

Other effects / acceptance of results:

First and second runs
All acceptance criteria were fulfilled for the positive and negative controls. The runs were therefore considered to be valid.
The results met the criteria of a positive response in both runs.

Interpretation of results:

study cannot be used for classification

Remarks:

The two major constituents of the test item (dodecylbenzenesulfonic acid/CAS 27176-87-0 and cyclohexanamine/CAS 108-91-8) are classified H314 “causes severe skin burns and eye damage” according to the classification provided by companies to ECHA in REACH registrations

Conclusions:

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor, though with caution due to the H314 classification ("causes severe skin burns and eye damage") of the two major constituents of the test item.

Executive summary:

The objective of this study was to evaluate the potential of the test item to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

The study was performed according to the international OECD guideline No.442D (except that no solubility test was performed since the test item was already formulated in water) and in compliance with the principles of Good Laboratory Practice.

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

Methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

Results

First run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered as validated.

 

This run was performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO and 1% water.

At these tested concentrations:

.            no precipitate/emulsion was observed in any wells at the end of the 48-hour treatment period at any tested concentrations,

.            a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations = 15.63 µM,

.            the corresponding IC₃₀ and IC₅₀ were calculated to be 11.58 and 12.73 µM, respectively,

.            a statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at 7.81 µM with an apparent overall dose-response relationship up to the cytotoxic concentrations,

.            the I_max was 2.16 and the calculated EC_1.5 was 4.27 µM.

Thus, the results met the criteria of a positive response

Second run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.

 

This run was performed using the following concentrations 1.43, 2.01, 2.84, 4.00, 5.64, 7.95, 11.2, 15.8, 22.3, 31.4, 44.3 and 62.5 µM in culture medium containing 1% DMSO and 1% water.

At these tested concentrations:

.            no precipitate/emulsion was observed in any wells at the end of the 48-hour treatment period at any tested concentrations,

.            a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations = 31.4 µM,

.            the corresponding IC₃₀ and IC₅₀ were calculated to be 26.99 and 28.31 µM, respectively,

.            statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at 15.8 and 22.3 µM with an apparent overall dose-response relationship up to the cytotoxic concentrations,

.            the I_max was 3.97 and the calculated EC_1.5 was 13.72 µM.

Thus, the results met the criteria of a positive response.

 

Global analysis from both runs:

The geometric means IC₃₀and IC₅₀of the twovalidated runs were 17.68 and 18.98 µM, respectively.

 

The evaluation criteria for a positive response were met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

Discussion

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay. However, it is worthy to note that the two major constituents of the test item are classified H314 "causes severe skin burns and eye damage" according to the classification provided by companies to ECHA inREACH registrations (information specified by the Sponsor in an email dated 31 March 2017 archived with the study files).

According to the EURL ECVAM Recommendation on the KeratinoSens assay for skin sensitization testing (European Commission - February 2014 (JRC 87551)), reactive chemicals that cause dermal corrosion/irritation without, however, being skin sensitizers, may lead to false positive results in the KeratinoSens test method.

Thus, in the context of an integrated approach to testing and assessment, this information should be considered and the result of this test be taken with caution.

Conclusion

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor, though with caution due to the H314 classification (“causes severe skin burns and eye damage”) of the two major constituents of the test item.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2017 - 14 March 2017

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))

Version / remarks:

04 February 2015

Deviations:

yes

Remarks:

Except that no solubility test was performed since the test item was already formulated in water (vehicle recommended by the OECD guideline). This deviation was considered not to have any impact on the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.
Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

Cysteine peptide (batch 111016HS_MHeW0117, supplier JPT Peptide Technologies GmbH)
Lysine peptide (batch 220114HSDWW0117, supplier JPT Peptide Technologies GmbH)

Vehicle used: milli-Q water. Since the test item supplied by the Sponsor was already formulated in water (vehicle recommended by the OECD guideline), no solubility test was performed and the vehicle used for the preparation of the test item dose formulation was water.

Positive control: cinnamaldehyde (CAS No. 104-55-2), batch No. MKBV4784V, supplied by Sigma Aldrich. Its molecular weight was 132.16 g/mol and the purity of the batch used was 98.9%. The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The physical aspect of the formulation was a colorless liquid. The formulation was used just after its preparation.

Criteria: Interpretation of results
The run was considered valid if the following criteria were fully met:
- the calibration curve should have a coefficient of determination (r2) >= 0.99,
- the mean peptide concentrations of the reference control A samples should be within ± 10% of the nominal concentration,
- the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
- for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100% with a SD < 14.9%,
- for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0% with a SD < 11.6%,
- the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.
The test item’s results were considered valid if the following criteria were fully met:
- the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
- the maximum SD for the test item replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

Parameter:

other: % depletion

Remarks:

lysine peptide

Value:

89.84

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: % depletion

Remarks:

cysteine peptide

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Parameter:

other: % mean depletion

Remarks:

Cysteine + Lysine

Value:

44.92

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The mean of cysteine and lysine %depletion = 44.92%. Accordingly, the test item was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive. The test item may have potential to cause skin sensitization.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in attached formula.
- for the cysteine peptide, the mean depletion value was 0.00%,
- for the lysine peptide, the mean depletion value was 89.84%.

It is to be noted that since phase separation (micelles) was observed at the end of the incubation with the cysteine peptide, the corresponding peptide depletion may be underestimated. Therefore, the mean depletion value of 0.00% cannot be drawn with sufficient confidence.

The mean of the percent cysteine and percent lysine depletions was equal to 44.92%. Accordingly, the test item was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

Conclusion: Under the experimental conditions of this study, the test item was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.

Interpretation of results:

other: considered positive in the DPRA assay

Remarks:

high peptide reactivity

Conclusions:

Under the experimental conditions of this study, the test item was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.

Executive summary:

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment. The study was performed according to the international OECD guideline No. 442C(except that no solubility test was performed since the test item was already formulated in water)and in compliance with the principles of Good Laboratory Practice.

 

Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

 

Results

The test item was dissolved at 100 mM in milli-Q water.

 

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in attached formula.

.            for the cysteine peptide, the mean depletion value was 0.00%,

.            for the lysine peptide, the mean depletion value was 89.84%.

 

The mean of the percent cysteine and percent lysine depletions was equal to 44.92%. Accordingly, the test item was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

 

Conclusion

Under the experimental conditions of this study, the test item was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 August 2017 to 15 August 2017

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:

OECD Guidelines for Testing of Chemicals No.429 Skin Sensitization: Local Lymph Node Assay (22 July 2010)

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42., “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L 142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD) Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 10 weeks old (age-matched, within one week)
Body weight range at starting: 20.1 – 22.2 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 21 days

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunneltubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.1 – 25.6°C
Relative humidity: 24 - 89 %
Ventilation: 15-20 air exchanges/hour

Food and feeding
Animals received ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance" (Batch number: 285 17890, Expiry date: 31 August 2017, and Batch number: 262 21592, Expiry date: 31 January 2018, produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch Number: 60162180010101, Expiry date: 05 August 2017, and Batch Number: 60170790050101, Expiry date: 20 March 2018, Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Vehicle:

other: 1% aqueous Pluronic® PE9200 (1% Pluronic)

Concentration:

Due to the physical characteristics of the test item (liquid gel), 100 % (undiluted) concentration was achievable. The formulation at 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) using 1% Pluronic as vehicle were suitable for the test.

No. of animals per dose:

4 animals per dose group

Details on study design:

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test I, was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of and 100% (undiluted) and 50% (w/v) in 1% Pluronic. Based on the observed ear swelling an additional test was performed. Preliminary Irritation/Toxicity Test II, using three doses (2 animals/dose) at test item concentrations of 25% (w/v), 10% (w/v) and 5% (w/v) in 1% Pluronic.
The preliminary experiments were conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100% (undiluted).
In the Preliminary Irritation / Toxicity Tests, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Tests, no mortality or signs of systemic toxicity were observed. No test item precipitate was observed on the ears of the animals in each experiment.
In the Preliminary Experiment I, alopecia around the ears were observed for one animal of the 100% (undiluted) dose group on Days 3-6 and other animal of the 100% (undiluted) dose group on Day 6. In the Preliminary Experiment II there were no indications of any irritancy at the site of application.
No marked body weight loss was observed in each experiment.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6.
In the Preliminary Experiment I the detected ear thickness values clearly indicated excessive local irritation (= 25 %) for both animals of the 100% (undiluted) and 50% (w/v) dose groups on Day 6 and for both animals (only right or left ear) of the 100% (undiluted) dose group on Day 3. The revealing ear punch weights were out of the historical control range for both animals of the 100% (undiluted) dose group, the increase was below the limit of positive (= 25 %) in the 100% (undiluted) dose group.
In the Preliminary Experiment II the detected ear thickness values clearly indicated excessive local irritation (= 25 %) for both animals of the 25% (w/v) dose groups on Day 3 and Day 6 and the detected ear thickness values indicated excessive local irritation (= 25 %) for one animal (only right ear) of the 25% (w/v) dose group on Day 3, however the other ear thickness values of the 25% (w/v), 10% (w/v) and 5% (w/v) dose groups were within the acceptable range. The revealing ear punch weights were within the historical control range in the 25% (w/v), 10% (w/v) and 5% (w/v) dose groups.
The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in all animals of the 100% (undiluted), 50% (w/v) and 25% (w/v) dose groups and they were normal in all animals of the 10% (w/v) and 5% (w/v) dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 100% (undiluted), 50% (w/v) and 25% (w/v) dose groups clearly exceeded the acceptable limit; therefore these concentrations were excluded from the examined concentration series of the main test. Based on these results, 10 % (w/v) dose is selected as top dose for the main test. Additionally, ear thickness of the experimental animals in the main test was measured by using a thickness gauge on Days 1, 3, 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determinations, which was performed after the animals are humanely killed.

Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Measurement of Ear Thickness
The ear thickness values of the experimental animals were determined by using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6 in the study.
Additional quantification of the ear thickness was also performed in the study by ear punch weight determination on Day 6 after the animals were euthanized.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of each mouse were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation, supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed.
Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The ß-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Positive control substance(s):

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

Statistics:

The use of the individual approach to calculate the SI made the use of a statistical analysis available. The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values.
Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by the Kolmogorow-Smirnow test. In the case of no normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using the Mann-Whitney U-test.

Positive control results:

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (1% Pluronic) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 6.9) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

Key result

Parameter:

SI

Value:

4.1

Test group / Remarks:

10 (w/v)% in 1% Pluronic

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

5 (w/v)% in 1% Pluronic

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

2.5 (w/v)% in 1% Pluronic

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. No test item precipitate was observed on the ears of the experimental animals. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT
Marked body weight loss (=5%) was observed for one animal of the negative control group, for both animals of the 2.5% (w/v) dose group and positive control group, however these changes were considered as individual variability. No treatment related effects were observed on the mean body weight changes.

EAR THICKNESS MEASUREMENT
The ear thickness values and revealing ear punch weights were within the historical control range. There was no indication of local irritation by visual assessment.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group and in 5% (w/v) and 2.5% (w/v) dose groups. Larger than normal lymph nodes were observed in the 10% (w/v) dose group and in the positive control group.

INTERPRETATION OF OBSERVATIONS
The test item was formulated in 1% Pluronic. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that Benzenesulfonic acid,4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine is a sensitizer (Figure 1). The size of lymph nodes was in good correlation with this conclusion. Based on the observed results the test item is classified as Category 1 (sub-category 1B) according to the GHS or CLP. The calculated EC3 value of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine is 8.0% (w/v).

Individual Body Weights for all Animals with Group Means

Animal Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight^*(g)	Change^#(%)
4430 4437 4441 4436	Negative (vehicle) control) 1% Pluronic     Mean	21.7 21.3 21.6 20.1 21.2	22.0 20.3 20.3 20.5 20.8	1.4 -4.7 -6.0 2.0 -1.8
4429 4442 4435 4440	Benzenesulfonic acid, 4-C10-13-sec-alkyl dervis., compds. with cyclohexylamine 10 (w/v)% in 1% Pluronic Mean	21.7 21.7 20.6 20.3 21.1	21.7 22.2 20.6 20.6 21.3	0.0 2.3 0.0 1.5 0.9
4434 4428 4445 4444	Benzenesulfonic acid, 4-C10-13-sec-alkyl dervis., compds. with cyclohexylamine 5 (w/v)% in 1% Pluronic Mean	22.2 21.1 20.9 20.1 21.1	22.9 21.4 21.1 20.3 21.4	3.2 1.4 1.0 -0.5 1.3
4433 4439 4427 4432	Benzenesulfonic acid, 4-C10-13-sec-alkyl dervis., compds. with cyclohexylamine 2.5 (w/v)% in 1% Pluronic Mean	21.6 21.6 20.3 20.3 21.0	20.2 20.1 20.8 19.8 20.2	-6.5 -6.9 2.5 -2.5 -3.4
4438 4443 4446 4431	Positive control 25 (w/v)% HCA in 1% Pluronic     Mean	22.1 21.2 20.3 20.3 21.0	23.9 19.8 19.1 20.7 20.9	8.1 -6.6 -5.9 2.0 -0.6

Notes:

*: terminal body weights were measured in Day 6

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DOM, DPN and Stimulation Index Values for all Groups

Test Group Name	Identity Number	Measured DPM	DPM	Number of lymph nodes	DPN	Group DPN	Stimulation Index
Background (5% (w/v) TCA)	-	32 31	-	-	-	-	-
Negative control (1% Pluronic)	1 2 3 4	184 197 187 192	152.5 165.5 155.5 160.5	2 2 2 2	76.3 82.8 77.8 80.3	79.3	1.0
Benzenesulfonic acid, 4-C10-13-sec-alkyl dervis., compds. with cyclohexylamine 10 (w/v)% in 1% Pluronic	5 6 7 8	477 884 467 879	445.5 852.5 435.5 847.5	2 2 2 2	222.8 426.3 217.8 423.8	322.6	4.1^*
Benzenesulfonic acid, 4-C10-13-sec-alkyl dervis., compds. with cyclohexylamine 5 (w/v)% in 1% Pluronic	9 10 11 12	253 168 382 195	221.5 136.5 350.5 163.5	2 2 2 2	110.8 68.3 175.3 81.8	109.0	1.4^NS
Benzenesulfonic acid, 4-C10-13-sec-alkyl dervis., compds. with cyclohexylamine 2.5 (w/v)% in 1% Pluronic	13 14 15 16	118 156 115 119	86.5 124.5 83.5 87.5	2 2 2 2	43.3 62.3 41.8 43.8	47.8	0.6^*
Positive control (25% HCA) in 1% Pluronic	17 18 19 20	986 1323 1033 1136	954.5 1291.5 1001.5 1104.5	2 2 2 2	477.3 645.8 500.8 552.3	544.0	6.9^*

Notes:

1. NS = Not Significant (Mann-Whitney U-Test versus negative control)

2. * = Significant (p<0.05 Mann-Whitney U-Test versus negative control)

Interpretation of results:

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

Conclusions:

In conclusion, under the conditions of the present assay, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine is 8.0% (w/v).
The following classification/labelling is triggered:
Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (sub-category 1B)

Executive summary:

The aim of this study was to determine the skin sensitisation potential of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine following dermal exposure in mice. The study was performed with vertebrate animals in order to confirm the positive results obtained with in vitro tests and if it is to determine the category.

 

In agreement with the Sponsor, 1% aqueous Pluronic® PE9200 (1% Pluronic) was used as vehicle for the test item in the study. Due to the physical characteristics of the test item (liquid gel), 100% (undiluted) concentration was achievable. The formulation at 50% (w/v), 25% (w/v), 10% (w/v), 5% (w/v) and 2.5% (w/v) using 1% Pluronic as vehicle were suitable for the test. As 1% Pluronic is one of the vehicles recommended by the relevant OECD guideline, it was selected for vehicle of the study.

 

The Preliminary Irritation/Toxicity Test I was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose) 100% (undiluted) and 50% (w/v) in 1% Pluronic. The Preliminary Irritation/Toxicity Test II was performed in CBA/CaOlaHsd mice using three doses (2 animals/dose) 25% (w/v), 10% (w/v), 5% (w/v) in 1% Pluronic. Based on the observations recorded in the preliminary tests, the 10% (w/v) dose was selected as top dose for the main test.

 

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine (formulated in 1% Pluronic) at 10% (w/v), 5 % (w/v) and 2.5% (w/v) concentrations,

- the negative control group received the vehicle (1% Pluronic),

- the positive control group received 25 % (w/v) HCA (dissolved in 1% Pluronic).

 

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

 

No mortality or signs of systemic toxicity were observed during the study. No test item precipitate was observed on the ears of the experimental animals. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight changes in the main study.

 

The stimulation index values were 4.1, 1.4 and 0.6 at concentrations of 10% (w/v), 5% (w/v) and 2.5% (w/v), respectively.

 

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine is 8.0% (w/v).

 

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (sub-category 1B).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Reporting format for defined approaches to testing and assessment based on multiple information sources

1 Summary

In vitro studies, performed onBenzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds with cyclohexylamine (2060540-82-9) showed that the substance is a skin sensitizer although some results have to be taken with caution. To confirm the in vitro results and determine the potency of skin sensitization, a skin sensitization test (Local Lymph Node Assay) was done, and concluded thatBenzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds with cyclohexylamineis a weak skin sensitizer.

 

2 General information

2.1 Identifier:

In order to evaluate the potential skin sensitizer properties and to not use animal tests, a strategy in two steps was done: at first with performing in vitro skin sensitization studies, and in the second step, a read-across with an in vivo skin sensitization study is proposed to determine the potency of skin sensitization of the registered substance.

 

2.2 Reference to main scientific papers:

-       ECHA.Guidance on Information Requirements and Chemical Safety Assessment.ChapterR.7a: Endpoint specific guidance. Version 6.0 – July 2017

-       ECHA.Guidance on Information Requirements and Chemical Safety Assessment.ChapterR.7c: Endpoint specific guidance. Version 2.0 – November 2014

-       OECD (2012) The Adverse Outcome Pathway for Skin Sensitization Initiated by Covalent Binding to Proteins, Part 1: Scientific Evidence, Series on Testing and Assessment No.168 (ENV/JM/MONO(2012)10/PART1), available at:http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2012)10/part1&doclanguage=en

 

3 Endpoint addressed

Skin sensitization

 

4 Definition of the purpose

Meeting the REACH information requirement for skin sensitization (Annex VII, 8.3) and the relevant classification and/or risk assessment obligations.

 

5 Rationale underlying the construction of the defined approach

 “Due to the complexity of the skin sensitization endpoint, a combination of alternative test methods (e.g.in silico, in chemico and in vitro) in a Weight-of-Evidence approach needs to be considered to increase confidence in the final assessment of skin sensitization, e.g. a combination of read-across and non-animal test methods can be useful in concluding on the assessment of skin sensitization. “ (ECHA Guidance R7a, 2017, page 302).

The rationale of the defined approach is based on the strategy described in the ECHA Guidance R7a (2017, Pages 304-307,Figure R.7.3–2 Testing and assessment strategy for evaluating the skin sensitization potential of substances).

 

Element	Information	Conclusion for the registered substance
Existing data on physico-chemical properties
1	Is the substance a strong acid (pH= 2.0) or base (pH= 11.5), corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature?	NO
Existing human data
2	Are there adequate existing human data, which provide evidence that the substance is a skin sensitizer?	NO
Existing animal data from sensitisation studies
3	Are there data from existing studies on skin sensitization in laboratory animals (LLNA, GPMT, or Buehler test, EU B.42, B.50, B.51 and B.6/OECD TGs 429, 442A, 442B and 406), which provide sound conclusive evidence that the substance is a sensitizer, or non-sensitizer?	NO
Existing (Q)SAR data and read-across
4	Do “read-across” from structurally and mechanistically related substances and/or do suitable (Q)SAR predictions reliably indicate skin sensitization potential or the absence thereof of the substance?	NO
Existing in chemico and in vitro data
5a	Is there evidence/hypothesis of dermal bioavailability based on physico-chemical, in silico, in vitro or in vivo data?	YES
5b	Has the substance demonstrated peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. OECD TG 442c)? (Key event 1 of the AOP), and/or Has the substance demonstrated activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. OECD TG 442d)?(Key event 2 of the AOP), and/or Has the substance demonstrated induction of the cell surface markers (CD54 and/or CD86) on monocytic cells in a validated in vitro test, e.g. h-CLAT? (Key event 3 of the AOP).	NO
5c	Are there data from (a) non-validated in vitro test(s), which provide evidence that the substance may be a skin sensitizer?	NO
Weight-of-Evidence analysis
6	The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-5) into account, is there sufficient information to meet the information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?	NO
Generation of new non-animal data
7a	Does the substance demonstrate peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. B. 59/OECD TG 442c)? (Key event 1 of the AOP)	YES
7b	Does the substance demonstrate activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. B.60/OECD TG 442d)? (Key event 2 of the AOP)	YES
7c	Does the substance demonstrate induction of the cell surface markers (CD54 and/or CD86) of monocytic cells in a validated in vitro test (e.g. h-CLAT)? (Key event 3 of the AOP)	N/A Test not performed.
7d	Is any additional testing/generation of data considered necessary in order to conclude on classification, or e.g. to explain the inconsistent data obtained in previous elements or to address the Key event 4 of the AOP (T-cell proliferation) with an in vitro test?	NO
Weight-of-Evidence analysis
8	The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-7) into account, is there sufficient information to meet the respective information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?	NO According to the generation of data through the performance of OECD 442c and 442d studies, the registered substance should be considered as skin sensitizer although some results have to betaken with caution. To confirm the in vitro results and determine the potency of skin sensitization, an additional in vivo test should be done.
Generation of new in vivo data for sensitization as a last resort (Annex VII to the REACH Regulation)
9	Does the substance demonstrate sensitizing properties in an EU/OECD adopted in vivo test, the LLNA (EU B.42/OECD TG 429)?	YES The registered substance has to be classified as weak skin sensitizer (Skin sens. 1B)

 

 

6 Description of the individual information sources used within the approach

 

Kerantinosens test

The objective of the test was to evaluate the potential of the test item, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine, to activate the Nrf2 transcription factor. This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferas gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. The study was performed according to the international OECD guideline No. 442D (except that no solubility

test was performed since the test item was already formulated in water) and in compliance with theprinciples of Good Laboratory Practice.

The first run was performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO and 1% water and the second one using the following concentrations 1.43, 2.01, 2.84, 4.00, 5.64, 7.95, 11.2, 15.8, 22.3, 31.4, 44.3 and 62.5 µM in culture medium containing 1% DMSO and 1% water.

At all these tested concentrations:

. no precipitate/emulsion was observed in any wells at the end of the 48-hour treatment period at any tested concentrations,

. a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations = 15.63 µM (1st run) or = 31.4 µM (2nd run),

. the corresponding IC30 and IC50 were calculated to be 11.58 and 12.73 µM (first run) and 26.99 and 28.31 µM (2nd run), respectively,

. a statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control

. the Imax were 2.16 and 3,97 (1st & 2nd runs, respectively) and the calculated EC1.5 4.27 and 13,72 µM (1st & 2nd runs, respectively)

. All acceptance criteria were met for the positive and negative controls.

Under the experimental conditions of this study, the test item, was positive in the KeratinoSens assay. However, it is worthy to note that the two major constituents of the test item (dodecylbenzenesulfonic acid/CAS 27176-87-0 and cyclohexanamine/CAS 108-91-8) are classified H314 "causes severe skin burns and eye damage"according to the classification provided by companies to ECHA in REACH registrations (informationspecified by the Sponsor in an email dated 31 March 2017 archived with the study files). According to the EURL ECVAM Recommendation on the KeratinoSens assay for skin sensitization testing (European Commission - February 2014 (JRC 87551)), reactive chemicals that cause dermal corrosion/irritation without, however, being skin sensitizers, may lead to false positive results in the KeratinoSens test method. Thus, in the context of an integrated approach to testing and assessment, this information should be considered and the result of this test be taken with caution.

DPRA

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

The study was performed according to the international OECD guideline No. 442C (except that no solubility test was performed since the test item was already formulated in water) and in compliance with the principles of Good Laboratory Practice.

The reactivity of the test item was evaluatedin chemicoby monitoring peptide depletion by HPLC with Ultra-Violet detection following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides.

The mean percent depletion values were calculated for each peptide and indicated:

.a mean depletion value of 0.00% for the cysteine peptide,

.a mean depletion value of 89.84% for the lysine peptide,

Therefore, the mean of the percent cysteine and percent lysine depletions was equal to 44.92%. Accordingly, the test item was considered to have a high peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization.

Under the experimental conditions of this study, the test item Benzenesulfonic acid, 4-C10-13-sec-alkyl

derivs., compds. with cyclohexylamine, was considered to have a high peptide reactivity. The test item is considered positive in the DPRA assay.

LLNA

The aim of this study was to determine the skin sensitisation potential of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine following dermal exposure in mice. The study was performed with vertebrate animals in order to confirm the positive results obtained with in vitro tests and if it is to determine the category.

1% aqueous Pluronic® PE9200 was used as vehicle for the test item in the study. Due to the physical characteristics of the test item (liquid gel), 100% (undiluted) concentration was achievable. The formulation at 50% (w/v), 25% (w/v), 10% (w/v), 5% (w/v) and 2.5% (w/v) using 1% Pluronic as vehicle were suitable for the test. Based on the observations recorded in the preliminary tests, the 10% (w/v) dose was selected as top dose for the main test. In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with

cyclohexylamine (formulated in 1% Pluronic) at 10% (w/v), 5 % (w/v) and 2.5% (w/v) concentrations,

 - the negative control group received the vehicle (1% Pluronic),

- the positive control group received 25 % (w/v) HCA (dissolved in 1% Pluronic).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. No test item precipitate was observed on the ears of the experimental animals. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight changes in the main study. The stimulation index values were 4.1, 1.4 and 0.6 at concentrations of 10% (w/v), 5% (w/v) and 2.5% (w/v), respectively.

The result of the positive control substancea-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine, tested in a suitable vehicle, was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with cyclohexylamine is 8.0% (w/v).

The following classification/labelling for skin sensitization is triggered according to Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (sub-category 1B).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the overall data and the criteria laid down in EU regulation (EC) n° 1272/2008/EC (CLP) and EU directive67/548/EEC, Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs.,compds. with cyclohexylamine has to be classified skin sensitizer category 1B (H317).