3-amino-N,N-dimethylpropan-1-aminium 2-C10-13-alkyl benzenesulfonate

Endpoint summary

Description of key information

There rae three in chemico/in vitro skin sensitisation tests, the DPRA, Keratinosens and h-CLAT. DPRA and Keratinosens were negative and h-CLAT positive. To clarify the skin sensitisation potential a Guinea pig maximisation tets was carried out, this showed no evidence of skin sensitisation 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:

Experimental start date 04 August 2016 Experimental completion date 06 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Cysteine prediction model only used due to ion pairing on column interfearing with Lysine results.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The reactivity and sensitization potential of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015).

Specific details on test material used for the study:

Sponsor’s identification Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine
Alternative name Witconate P-1460
Product name ACAR 16015
Batch No. 2629-89-10
CAS No. 1093628-27-3
Purity 98.6%
Molecular weight 416.5
Appearance Amber paste
Expiry/retest date 03 April 2019
Storage conditions Room temperature in the dark

Details on the study design:

Peptide and Positive Control
Synthetic peptide containing Cysteine
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Stated purity: 95% (by HPLC)
Molecular Weight: 751.5 g/mol
Supplier: AnaSpec
Storage conditions: Frozen, -20°C
Expiry/retest date: 5 years.

Synthetic peptide containing Lysine
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Stated purity: 90-95%
Molecular Weight: 776 g/mol
Supplier: AnaSpec
Storage conditions: Frozen, -20°C
Expiry/retest date: 5 years.
Cinnamic Aldehyde (Positive control)
Batch number: MKBR2427V
Stated purity: >95%
Molecular Weight: 132.16 g/mol
Supplier: SAFC
Storage conditions: Room temperature
Expiry/retest date: February 2019

Apparatus
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 decimal places
General laboratory apparatus and glassware.

Analytical Procedure
Reagents
Acetonitrile (ACN): HPLC gradient grade
Methanol (MeOH): HPLC grade
Trifluoroacetic acid (TFA): 99% Pure
Water: Deionised reverse osmosis
Ammonium Acetate: Analytical reagent
Sodium Phosphate, monobasic: Analytical reagent
Sodium Phosphate, dibasic: Analytical reagent
Ammonium Hydroxide: Analytical reagent
100 mM Phosphate buffer, pH 7.5: In house preparation
100 mM Ammonium Acetate buffer, pH 10.2: In house preparation
HPLC Mobile Phase A: 0.1% v/v TFA in Water, in house preparation
HPLC Mobile Phase B: 0.085% v/v TFA in ACN, in house preparation

Assessment of Test Item Solubility
The solubility of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) in acetonitrile and methanol was assessed at a concentration of 100 mM.

Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

Preparation of Stability Controls and Precision Controls
Stability controls and precision controls of both peptides were prepared at a concentration of 0.5 mM.

Preparation of Positive Control Solution
The positive control chemical (Cinnamic aldehyde) was prepared at a concentration of 100 mM in both acetonitrile and methanol.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Methanol solutions of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) and the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) or the positive control.

For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Methanol solutions of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) and the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of the peptides containing Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) and the associated positive control was quantified by HPLC using UV detection as detailed in the chromatographic section.

Instrumentation Parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30°C
Sample temperature: 25°C
Mobile Phase (MP) A: 0.1% TFA in Water
Mobile Phase (MP) B: 0.085% TFA in ACN

Gradient: Time (minutes) MPA(%) MPB (%)

0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
Flow rate: 0.35 mL/minute
Stroke volume 25 µL
Detector wavelength: UV, 220 nm
Injection volume: 2 µL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine) 11 minutes
Approximate retention time (Lysine) 7 minutes

Calculations
The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% Peptide depletion = 100 - ((Peptide peak area in replicate depletion samples (x 100)) / Mean Peptide peak area of reference control samples B or C)
For clarification, control samples B were used for calculation of positive controls and control samples C were used for calculation of depletion samples.

Major Computerized Systems
Chromatography data handling: Waters Empower
Sample registry system: Envigo
Test substance management: Pristima, Xybion Medical Systems Corporation
Version numbers of the systems are maintained by Envigo.

Positive control results:

Not applicable

Key result

Run / experiment:

other: Results based on Cysteine assessment model as Lysine peptide results are not due to ion pairing interaction on the HPLC column

Parameter:

other: peptide binding to synthetic peptides

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no indication of skin sensitisation

Solubility Assessment

The solubility of the Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) in acetonitrile at a nominal concentration of 100 mM was not confirmed, solubility was confirmed in methanol at a nominal concentration of 100 mM.

Reactivity Assessment

The DPRA prediction and the reactivity of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) based on the Cysteine peptide depletion and the individual depletion values in the Cysteine peptide and the Lysine peptide is presented inTable 1. The Lysine peptide result was confirmed to be a false positive due to ion-pairing interactions, with an extra late running peak seen eluting in the chromatogram that when summed with the Lysine peptide peak areathe total peak area matched that of the positive control. As the result generated when using the Lysine containing synthetic peptides was affected by these ion-pairing interactions, the Lysine result has been excluded and only the Cysteine result is used for classification.

All analytical acceptance criteria for each peptide run were met:

 

		Peptide		Linearity		Positive control depletion (%)		Reference controls		Test item
Acceptance criteria		Cysteine		>0.99		60.8-100 (CV <14.9%)		0.45-0.55 mM (CV <15%)		CV <14.9%
		Lysine		>0.99		40.2-69.0 (CV <11.6%)		0.45-0.55 mM (CV <15%)		CV<11.6%
Achieved results		Cysteine		>0.999		73.1 (CV, 0.01%, n=3)		B: 0.511 mM (CV 0.61%, n=6) C: 0.507 mM (CV 0.67%, n=3)		CV 0.27%
		Lysine		>0.999		58.0 (CV, 4.31%, n=3)		B: 0.511 mM (CV 1.33%, n=6) C: 0.509 mM (CV 0.84%, n=3)		CV 3.32%

CV         Coefficient of Variation

The depletion of peptide in the presence of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) was:

	Mean peak area of reference controls(µV.sec)	Mean peak area of test item(µV.sec)	Mean Depletion (%)
Cysteine	Control B (ACN): 867260 (n=6) Control C (MeOH): 861110 (n=6)	856140 (n=3)	0.577
Lysine	Control B (ACN): 745970 (n=6) Control C (MeOH): 743560 (n=6)	112430 (n=3)	84.9^1

¹       The mean Lysine result has been excluded from calculation due to the false positive result due to ion-pair interactions with the HPLC column.

And applying the following Cysteine 1:10 depletion model (below), reactivity as minimal and the DPRA prediction is negative and Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) is therefore a non skin sensitizer. 

Cysteine (Cys) % depletion	Reactivity Class	DPRA Prediction
0%≤ Cys% depletion ≤13.89%	No or minimal reactivity	Negative
13.89%< Cys % depletion ≤23.09%	Low reactivity	Positive
23.09%< Cys % depletion ≤98.24%	Moderate reactivity
98.24%< Cys % depletion ≤100%	High reactivity

 

No co-elution occurred in either assay.  

Overall Achieved Depletion Values

Test item		Cysteine peptide depletion (%)		Lysine peptide depletion (%)		Cysteine peptide depletion (%)		Reactivity class		DPRA prediction
Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015)		0.577		84.9^1		0.577		No or minimal		Negative

¹             The mean Lysine result has been excluded from calculation due to the false positive result due to ion-pair interactions with the HPLC column.

 

 Individual Achieved Depletion Values

 Cysteine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion (%)	Mean Depletion (%)	CV (%)
Positive control	233643	103.67	73.1^2	73.1	0.01
	233670	103.68	73.1^2
	233683	103.69	73.1^2
Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015)	857223	379.43	0.451^3	0.577	0.27
	853515	377.79	0.882^3
	857671	379.63	0.339^3

CV      Coefficient of variation

¹                Samples prepared at a concentration of 376 µg/mL (0.5 mM)

²                Calculated against a mean control peak area of 867260 µV.sec(Control B, n=6)

³          Calculated against a mean control peak area of 861110 µV.sec(Control C, n=3)

 Lysine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion(%)	Mean Depletion (%)	CV (%)
Positive control	300390	159.62	59.7^2	58.0	4.31
	327364	173.97	56.1^2
	312596	166.11	58.1^2
Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015)	109938	58.330	85.2^3	84.9	3.32
	110630	58.698	85.1^3
	116726	61.940	84.3^3

CV      Coefficient of variation

¹                Samples prepared at a concentration of 388 µg/mL (0.5 mM)

²          Calculated against a mean control peak area of 745970 µV.sec(Control B, n=6)

³          Calculated against a mean control peak area of 743560 µV.sec(Control C, n=3)

 

Interpretation of results:

GHS criteria not met

Conclusions:

Solutions of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in Cysteine containing synthetic peptides. Solutions of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) analyzed by the validated method in Lysine containing synthetic peptides appeared to show the test item to be a strong skin sensitizer. This was investigated further and was confirmed to be a false positive due to ion-pairing effects between the test item and the HPLC column, as a result the Lysine result has been excluded and only the Cysteine result is reported
The Cysteine result places Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) in the reactivity class of “no or minimal reactivity” and therefore it is predicted to be a non skin sensitizer.

Executive summary:

This purpose of this study (based on the OECD guideline for the testing of chemicals,In chemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015). 

Solutions of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in Cysteine containing synthetic peptides.

Solutions of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) analyzed by the validated method in Lysine containing synthetic peptides appeared to show the test item to be a strong skin sensitizer. This was considered to be a false positive due to the presence of an extra peak in the chromatogram eluting at approximately 13 minutes, when the peak areas of the Lysine peptide peak and this extra peak were summed, the total peak area matched that of the positive controls in the Lysine assay. It was considered that this could be the result of ion-pairing interactions with the HPLC column used in the validated method. An investigation confirmed the false positive result was due to ion-pairing effects between the test item and the HPLC column used in the validated method. As the result generated when using the Lysine containing synthetic peptides was affected by these ion-pairing interactions, the Lysine result has been excluded and only the Cysteine result is used for classification.

The Cysteine result places Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) in the reactivity class of “no or minimal reactivity” and therefore it is predicted to be a non skin sensitizer. 

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Treatment with test item: 29 and 31/08/16 Euthanasia and necorpsy: 07/02/17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The default animal study for skin sesnitisation, in the ECHA Guidelines, is the Local Lymph Node Assay (LLNA)OECD429, however it is accepted in the OECD guideline for the LLNA that it has some limitations, as follows: “Despite the advantages of the LLNA over TG 406, it should be recognised that there are certain limitations that may necessitate the use of TG 406 (13) (e.g. false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants [such as some surfactant type chemicals] (19) (20), or solubility of the test substance)”.

As Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-Propanediamine is a surfactant with some moderate irritant properties, there is a strong possibility of a false positive result from the LLNA, therefore the Guinea Pig Maximisation test (OECD406) test was carried out as this can be expected to give a result which is more relevant to the human hazard.

Specific details on test material used for the study:

• Sponsor’s identification: ACAR16015 Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamine
• Batch No.: 2629-89-10 • Cas No.: 1093628-27-3
• Date received: 01 June 2016 • Storage: room temperature, darkness
• Chemical name: Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamine
• Container: smoked glass flask (n=1) • Form: solid (paste)
• Quantity: 396.90 g (container + content) • Colour: yellowish
• Production date: 04 April 2016 • Expiry date: 03 April 2019
• Composition: 98.6% of Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamine

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

- Housing
The animals were housed in groups of 3 at the maximum in polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted with a stainless steel lid with a feeding device and drinking device of 500 mL.

The temperature and relative humidity of the main test were controlled to remain within target ranges of 19°C to 25°C and 30% to 70%, respectively.

The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.


Food and drink

The drinking water (tap water from public distribution system) and food (ENVIGO, 2040C) were supplied ad libitum. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).

Preparation of animals

Fifteen female albino guinea pigs of Dunkin-Hartley strain were supplied by Envigo (Kreuzelweg 53, 5961 NM HORST - The Netherlands). At the beginning of the main test, the animals were 4 or 5 weeks old. The animals were nulliparous and non-pregnant.

Prior to the test, the animals were kept for a minimum acclimatization period of 5 days, under stabling and nutritional conditions identical to those of the test.

Before the experimentation process, they were identified individually by marking with picric acid and by means of a numbered ring on the edge of one ear.

The animals were carefully shorn before each test item application:
- On the inter-scapular zone for the induction phase,
- On the dorso-lumbar zone for the challenge phase.

At least 3 hours before the first reading (challenge phase) they were shorn a second time in this dorso-lumbar zone.

The animals were weighed at the beginning of the test, after the second induction and at the end of the test.

Animal welfare
The standard study plan related to this study has been approved by the registered Ethics Committee No. 76.

The study was performed in accordance with the guidelines regarding the care and use of animals for experimental procedures:
- the European Communities Council Directive 2010/63/EU of 22 September 2010,
- the French Decree No. 2013-118 of 01 February 2013.

The animals were provided with suitable environmental enrichment (Tunnel).

The study was designed and was conducted to cause the minimum of suffering or distress to the animals, according to the guidelines and to our internal animal welfare’s procedure.

At the end of the test, the animals were euthanized with sodium pentobarbital (Dolethal®).

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

GROUP 1 (control):
• 2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
• 2 ID: physiological saline
• 2 ID: a mixture with equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and physiological saline

Day(s)/duration:

7

Adequacy of induction:

other: - Induction phase Group 1: No cutaneous reaction was noted during induction phase

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

GROUP 2 (Treated):
• 2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
• 2 ID: test item at 0.2% in physiological saline
• 2 ID: a test mixture in equal volumes v/v :
- Freund’s Complete Adjuvant at 50% and the test item at 0.4% in physiological saline

Day(s)/duration:

7

Adequacy of induction:

other: 24 hours after the first induction, moderate erythema was noted in all animals (10/10). Discrete erythema associated with dryness of the skin was noted in all animals (10/10) after the second induction

Route:

other: 2nd Topical induction

Vehicle:

physiological saline

Concentration / amount:

A topical application under occlusive dressing (25mm x 25mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 hours was performed on the injection sites of each animal.

GROUP 1 (control): 0.5 mL of distilled water.
GROUP 2 (treated): 0.5 mL of the test item at 2% in distilled water.

Day(s)/duration:

2

Adequacy of induction:

other: The animals of both groups were left for 11 days.

No.:

#20

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

- 1 sample cup containing the test item diluted at 4% (MNIC) and 1 sample cup containing the test item diluted at 2% in distilled water (1/2 MNIC).

Day(s)/duration:

1 day exposure, then 48-hours observation

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Main study

GROUP 1 (negative control): 5 female guinea pigs identified n° C9036 to C9040

GROUP 2 (treated): 10 female guinea pigs identified n° C9041 to C9050

Details on study design:

- Preliminary studies

Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC)

This test was conducted for the purpose of defining a MNNC of the test item which, on intradermal injection during the induction phase, does not risk causing too great a lesion (non-necrotizing concentration), should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation.

Two animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: diluted at 50%, 30%, 20% and 10% in physiological saline view to determine the MNNC.

Due to necrosis following the injections, the same animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 new concentrations: diluted at 5%, 2%, 1% and 0.5% in physical saline in view to determine the MNNC.

Due to necrosis following the new injections, the same animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 new concentrations: diluted at 0.2%, 0.1%, 0.05% and 0.02% in physical saline in view to determine the MNNC.

A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after the injections.


Determination by topical application of the Pre-Maximal Non Irritant Concentration
(Pre-MNIC)

This test, which allowed evaluating the irritancy potential of the test item, defined whether an application of sodium lauryl sulfate would be needed during topical induction phase.

The test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: undiluted (100%), diluted at 75%, 50%, and 25% in distilled water.

After the removal of the occlusive dressing, the treated areas were rinsed with distilled water.

Due to intense erythema and scab following the application of the test item, the test item was applied on 2 other animals, at 4 new different concentrations: diluted at 10%, 5%, 2% and 1% in distilled water.

A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.


Determination by topical application of the Maximal Non Irritant Concentration (MNIC)
This test was carried out for the purpose of determining the MNIC of the test item without risk of an irritant effect during the challenge phase.

Three guinea pigs were treated according to the same treatment as animals from GROUP 1 (control) for the induction phase (i.e. physiological saline and distilled water).

During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations: diluted at 4%, 2%, 1% and 0.5% in distilled water.

A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressing.

Challenge controls:

Day 22
The experimental procedure of this phase was identical for both groups GROUP 1 (Control) and GROUP 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application, under occlusive dressing, was performed during 24 hours:

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamaldehyde CAS No 101-86-0, 12.5% and 6.25% run every 6 months to confirm sensitivity of the rabbits

Positive control results:

The results of the 3 last positive control groups (Reference substance: alpha-Hexylcinnamaldehyde (Tests 31-33) carried out in order to assess the sensitivity of the strain of guinea pig used at these laboratories to a known sensitiser are presented in the attached Appendix 2. They confirmed the sensitivity of the rabbits used in the laboratory to detect this refernce skin sensitiser.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

4% and 2%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

In the treated groups (treatment doses of 4% and 2%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

4% and 2%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

In the treated groups (treatment doses of 2% and 4%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.

Remarks on result:

no indication of skin sensitisation

- Preliminary studies

 

-MNNC determination:

24 hours after the injections,necrosis was observed in the animals at the tested concentrations of 50%, 30%, 20%, 10%, 5%, 2% and 1% and slight necrosis was observed at the tested concentration of 0.5%.

No cutaneous reaction was noted at the tested concentrations of 0.2%, 0.1%, 0.05% and 0.02%.

The first induction of the Group 2 was carried out by intradermal injection at the maximal non necrosing concentration of 0.2% (Table 1 attached).

 

-Pre MNIC determination:

24 hours after the removal of the occlusive dressings, a death occurred (1/2) and intense erythema was observed at the tested concentration of 100%, 75%, 50% and 25% in the other animal.

Due to the results, another run was performed with two other animals. 24 hours after the removal of the occlusive dressings,intense erythema was noted at the tested concentrations of 10% and 5%. No cutaneous reaction was noted at the tested concentrationsof 2% and 1%(Table 2).

 

 

In view of these results, the concentration selected was 2% for the 2^ndinduction of the Group 2 and the MNIC determination began at the concentration of 4%.

 

-MNIC determination:

24 and 48 hours after the removal of the occlusive dressings, no cutaneous reaction was noted whatever the tested concentration (Table 3).

 

In view of this result, the concentrations selected were 4% (MNIC) and 2% (1/2 MNIC).

 

Main study

 

-Induction phase Group 2:

24 hours after the first induction, moderate erythema was noted in all animals (10/10). Discrete erythema associated with dryness of the skin was noted in all animals (10/10) after the second induction (Appendix 4 attached).

 

-Induction phase Group 1:

No cutaneous reaction was noted during induction phase (Appendix 4 attached).

 

-Challenge phase Groups 1 & 2:

Overall results of the challenge phase with the test item (readings at 24 and 48 hours) are givenin Table 4 (attached)

Individual scores of macroscopic evaluations performed during challenge phase with the test item are given inTable 5 (attached).

 

In the treated group (treatment dose of 4%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.

In the control group (associated with the treatment dose of 4%), nomacroscopiccutaneous intolerance reactions were recorded after the challenge phase.

 

In the treated group (treatment dose of 2%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.

In the control group (associated with the treatment dose of 2%), nomacroscopiccutaneous intolerance reactions were recorded after the challenge phase.

 

Body Weights

The weight gain of control animals (Group 1) and treated animals (Group 2) is presented inTables 6 and 7,respectively (attached).

 

No abnormality was recorded in the body weight gain of both groups.

  

Mortality

 

No mortality was registered during the main test.

Interpretation of results:

GHS criteria not met

Conclusions:

In view of these results, under these experimental conditions, the test item ACAR16015 Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamine does not have to be classified in category 1 as a skin sensitizer, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.
No signal word or hazard statement is required.

Executive summary:

SUMMARY AND CONCLUSION OF THE STUDY

 

 The aim ohe study was to evaluate the possible allergenic activity of the test item after intradermal and topical administration in guinea pigs.

 

According the results of the pretests, the induction phase (intradermic injection at 0.2% and topical application at 2%) was conducted with the test item ACAR16015 Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamineto 10 Guinea pigsand a 11-day rest phase. The challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test item diluted at 4% and 2% in distilled water. The experimental protocol was established according to the Magnusson and Kligman method (J. Invest. Dermatol. 1969. 52, 268-276) and in accordance with O.E.C.D. Test Guideline No.406 of July 17^th, 1992, and the test method B.6 of council regulation No. 440/2008.

In the treated group (treatment dose of 4%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.

In the control group (associated with the treatment dose of 4%), no macroscopiccutaneous intolerance reactions were recorded after the challenge phase.

 

In the treated group (treatment dose of 2%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.

In the control group (associated with the treatment dose of 2%), no macroscopiccutaneous intolerance reactions were recorded after the challenge phase.

 

 

In conclusion, in view of these results, under these experimental conditions, the test itemACAR16015 Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediaminedoes not have to be classified in category 1as a skin sensitizer,in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

No signal word or hazard statement is required.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Star of study 21st November 2016 - Final report12th January 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:

KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This in vitro method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to induce the Keap1-Nrf2-ARE signalling pathway by quantifying the luciferase gene expression using luminescence detection.

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

Chemical Name: Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine
CAS No.: 1093628-27-3
Batch No.: 2629-89-10
Physical State: paste
Colour: amber
Molecular Weight: 416.5 g/mol
Purity: 98.6 g/mL
Storage Conditions: room temperature, protected from light
Stability: stable
Expiry Date: 03 April 2019

Details on the study design:

Preparation of the Test Item
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%;
Applichem; Lot No.: 0000978834). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a
4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells.
Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Controls
A blank, a negative control and a positive control were set up in parallel in order to confirm the
validity of the test.
Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO (Applichem; Lot No.: 0000978834) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; (Sigma; Lot No.: MKBS2662V) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Cell line
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded
in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 06 (experiment 1), P 06 (experiment 2)) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 +/- 1°C and 5% CO2. For test material exposure, cells were cultured in medium for test item exposure.

Composition of Media
Maintenance Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-108, Lot No.: 1786951) with 1.0 g/L D-glucose and Na-Pyruvate. The medium will be supplemented with the following components:
- 10% fetal bovine calf serum (Biochrome, Cat. No.: S0615, Lot No.: 0367A)
- 1% geneticin (final concentration: 500 µg/mL) (Gibco Life Science, Cat. No. 10131027, Lot No.: 1717097)

Assay Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-108, Lot No.: 1801714) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum (Biochrome, Cat. No.: S0615, Lot No.: 0391E)

Test Item Exposure Medium
Dulbecco’s Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-108, Lot No.: 1801714) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 1% fetal bovine calf serum (Biochrome, Cat. No.: S0615, Lot No.: 0391E)

Luciferase Assay System
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacturer’s manual. Luciferase Assay System 10-Pack
The kit (Promega, Cat. No.: E1501, Lot No.: 0000211803) consisted of the following components relevant for this study:
- 10 x Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer
Thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.

Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000199324) consists of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:4 with dist. water (Sigma; Lot No.: RNBF3331)

Further Reagents
MTT solution
- MTT (CAS No.: 298-93-1; Sigma; Lot No.: MKBR65) stock solution: 5 mg/mL MTT in DPBS (Gibco Life Science; Lot No.: 1813255)
SDS solution:
- 10% (w/v) sodium dodecyl sulfate (SDS; CAS No.: 151-21-3; AppliChem; Lot No.: 40015277) in dist. water (VWR; Lot No.: 16G054105)

DPBS:
DPBS solution (without Ca2+/Mg2+) (Gibco Life Science; Lot No.: 1813255)

Dose Groups
1. Negative Control: DMSO: 1% (v/v) in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates.

Experimental Procedure
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-
well plate (flat bottom) for the determination of the cell viability.

After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.

All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of
light.

Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and OD was measured at λ = 600 nm.

Data Analysis
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consists of three replicates for every concentration step of the test item and the positive control. In case of
discordant results a third independent run should be performed.

The following parameters were calculated:

Calculation of Cell Viability
Cell viability was calculated according to equation 1.
Cell Viability[%] = (Vsample-Vblank) x100
(Vsolvent-Vblank)

Vsample = MTT absorbance reading in the test chemical well
Vblank = MTT absorbance reading in the blank well containing no cells and no treatment
Vsolvent = average MTT absorbance reading in the wells containing cells and solvent (negative) control

Calculation of the Maximal Induction of the Luciferase Activity (Imax)
The maximal average fold induction of luciferase activity (Imax) value observed at any concentration
of the test item was calculated according to equation 2.

Fold induction = (Lsample-Lblank)
(Lsolvent-Lblank)

Lsample = luminescence reading in the test chemical well
Lblank = luminescence reading in the blank well containing no cells and no treatment
Lsolvent = luminescence reading in the wells containing cells and solvent (negative) control

Calculation of the EC1.5
The EC1.5 will be calculated by linear extrapolation according to equation 3, and the overall EC1.5 was calculated as the geometric mean of the individual repetitions.

EC1.5 = (Cb - Ca) x (1.5-Ia) + Ca
(Ib-Ia )
Ca = lowest concentration in µM with >1.5 fold induction
Cb = highest concentration in µM with <1.5 fold induction
Ia = fold-induction measured at the lowest concentration with >1.5 fold induction (mean of three replicate wells)
Ib = fold-induction measured at the highest concentration with <1.5 fold induction (mean of three replicate wells)

Calculation of IC50 and IC30
The IC50 and IC30 was calculated by linear extrapolation according to equation 4 and the overall IC50 and IC30 was calculated as the geometric mean of the individual repetitions.

ICx = (Cb - Ca) x ( (100-x))-Va) + Ca
( Vb-Va )

X = % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca = the lowest concentration in µM with >X% reduction in cell viability
Cb = highest concentration in µM with Va = % viability at the lowest concentration with >X% reduction in cell viability
Vb = % viability at the highest concentration with
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.

The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction on cellular viability at the EC1.5 determining concentration.

Prediction Model:
The test item is considered positive in accordance with UN GHS “Category 1” if the following
conditions were met in at least two independently prepared test repetitions.



- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative
control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is < 1000 µg/mL
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition will be considered inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM were considered as inconclusive.

Acceptance Criteria:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition consisting of 6 wells.

Key result

Run / experiment:

other: Based on Experiment 1 and 2

Parameter:

other: Luciferase activity increase - No relevant dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Cytotoxicity

Table 1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Fold Induction
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100.0	100.0	100.0	0.0
Positive Control	4.00	106.5	97.6	102.1	6.2
	8.00	120.0	94.6	107.3	18.0
	16.00	127.0	113.1	120.0	9.8
	32.00	125.9	112.4	119.1	9.5
	64.00	147.7	120.5	134.1	19.2
Test Item	0.98	107.6	100.4	104.0	5.1
	1.95	112.5	79.5	96.0	23.4
	3.91	120.1	86.5	103.3	23.7
	7.81	110.5	110.5	110.5	0.1
	15.63	170.5	146.1	158.3	17.3
	31.25	17.2	0.1	8.7	12.1
	62.50	-0.1	0.2	0.0	0.2
	125.00	-0.6	0.4	-0.1	0.7
	250.00	0.0	0.5	0.2	0.4
	500.00	-0.2	0.6	0.2	0.5
	1000.00	-0.6	0.2	-0.2	0.6
	2000.00	-0.4	0.6	0.1	0.7

Table 2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [PM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.15	1.02	1.15	1.11	0.08
	8.00	1.16	1.31	1.28	1.25	0.08
	16.00	1.57	1.40	1.52	1.50	0.09
	32.00	1.66	1.57	1.44	1.55	0.11	*
	64.00	2.96	3.00	2.49	2.82	0.29	*
Test Item	0.98	0.89	0.95	0.87	0.91	0.04
	1.95	0.99	0.96	0.88	0.94	0.06
	3.91	0.97	1.03	0.93	0.98	0.05
	7.81	0.93	0.99	0.89	0.94	0.05
	15.63	1.64	1.58	1.19	1.47	0.25
	31.25	1.24	0.00	3.62	1.62	1.84
	62.50	0.00	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

* = significant induction according to Student’s T-test, p<0.05

Table 3: Induction of Luciferase Activity Experiment 2

Experiment 1	Concentration [PM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	0.94	0.94	1.27	1.05	0.19
	8.00	1.43	1.09	1.21	1.24	0.17
	16.00	1.28	1.28	1.54	1.36	0.15
	32.00	1.65	1.36	1.81	1.60	0.23	*
	64.00	2.20	2.03	2.91	2.38	0.47	*
Test Item	0.98	1.18	0.77	1.10	1.02	0.22
	1.95	0.87	0.80	0.91	0.86	0.06
	3.91	0.89	0.74	0.94	0.86	0.10
	7.81	0.91	0.87	1.09	0.96	0.11
	15.63	1.29	0.99	1.67	1.32	0.34
	31.25	3.93	5.03	5.15	4.70	0.67	*
	62.50	0.00	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

* = significant induction according to Student’s T-test, p<0.05

Table 4: Induction of Luciferase Activity – Overall Induction

=	Concentration [PM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.11	1.05	1.08	0.04
	8.00	1.25	1.24	1.25	0.01
	16.00	1.50	1.36	1.43	0.09
	32.00	1.55	1.60	1.58	0.04	*
	64.00	2.82	2.38	2.60	0.31	*
Test Item	0.98	0.91	1.02	0.96	0.08
	1.95	0.94	0.86	0.90	0.06
	3.91	0.98	0.86	0.92	0.08
	7.81	0.94	0.96	0.95	0.01
	15.63	1.47	1.32	1.39	0.11
	31.25	1.62	4.70	3.16	2.18
	62.50	0.00	0.00	0.00	0.00
	125.00	0.00	0.00	0.00	0.00
	250.00	0.00	0.00	0.00	0.00
	500.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00

* = significant induction according to Student’s T-test, p<0.05

Table 5: Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	18.75	16.47	17.59	1.59
Imax	1.62	4.70	3.16	2.18
IC30[µM]	25.87	23.77	24.82	1.49
IC50[µM]	27.91	25.91	26.91	1.41

 

Table 6: Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	<20%	12.5	pass	19.3	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	>1	2.0	pass	2.0	pass
EC1.5 PC	7 < x < 30µM	17.20	pass	25.05	pass
Induction PC at 64µM	2.00 < x < 8.00	2.82	pass	2.38	pass

 

Table 7: Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	<20%	11.0	2.6	29
No. of positive control concentration steps with significant luciferase activity induction >1.5	>1	2.4	0.5	29
EC1.5 PC	7 < x < 30µM	17.66	5.20	29
Induction PC at 64µM	2.00 < x < 8.00	3.81	1.02	29

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in thetransgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

Results

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line

KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

In the present study Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1 -dimethyl-1,3-propanediamine was dissolved in DMSO.

Based on a molecular weight of 416.5 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable solution was formed. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 61.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.62 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 17.2%. This concentration was the only one with a luciferase induction >1.5. The calculated EC1.5 was < 1000 µM (18.72 µM).

In the second experiment, a max luciferase activity (Imax) induction of 4.70 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 0.1%. This concentration was the only one with a significant luciferase induction >1.5. The calculated EC1.5 was < 1000 µM (16.47 µM).

No relevant dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Conclusion

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study started 3rd November 2016 - Final Report 6th March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E: In-Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st,
2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

Specific details on test material used for the study:

Name: Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds.
with N,N-dimethyl-1, 3-propanediamine
Chemical Name: Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds.
with N,N-dimethyl-1, 3-propanediamine
CAS No.: 1093628-27-3
Batch No.: 2629-89-10
Physical State: amber paste
Colour: amber
Molecular Weight: 416.5 g/mol
pH value: 6 - 7
Storage Conditions: room temperature, protected from light
Stability: stable
Expiry Date: 03 April 2019

Details on the study design:

Preparation of the Test Item
The test item was freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%, Lot No.: 0000803731) at a concentration of 500 mg/mL, respectively. Test items of limited solubility were dissolved at the highest soluble concentration in DMSO. Stock solutions were prepared diluting the highest soluble concentration seven times with a constant dilution factor of 1:2.

The working stock solutions were prepared diluting each stock solution 250 times with cell culture medium.

The working stock solutions were applied to the cells by adding equal volumes of each solutions to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Controls
Solvent controls and a positive control were set up in parallel in order to confirm the validity of the test.

Solvent Controls
The solvent control was set up depending on the appropriate solvent previously determined (see chapter 10.6.2).

For chemicals solubilized in either cell culture medium or 0.9% NaCl, cell culture medium was the solvent control.

For chemicals solubilized in DMSO, DMSO will be the solvent control.

The solvent controls will be diluted according to the procedure described for the test item in the Experimental proceedures chapter, resulting in a final concentration of 1% (v/v) for 0.9% NaCl and 0.2% (v/v) for DMSO.

Positive Control
2,4-dinitrochlorobenzene (DNCB; CAS No.: 97-00-7, purity ≥ 99 %, Lot No.: BCBP8259V) at a final concentration of 4 µg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described
for the test item in experimental proceedures chapter, resulting in a final DMSO concentration of 0.2% (v/v).

Test System
FACS
FACS: BD Canto II
Software BD FACS DIVA 6.0

Cell line
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used.

Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum (FBS, FBS, Biochrome, Cat No.: S 0615, Lot: 0391E), 25 mM HEPES (Gibco Life Science, Cat No.:15630-056 Lot.: 1788128), L-glutamine (Gibco Life Science, Cat. No.: 25030-081, Lot: 1801687), 2-mercaptoethanol (Gibco Life Science, Cat No.: 21985-023, Lot: 1779219) and penicillin/streptomycin (Gibco Life Science, Cat. No.: 15240-062, Lot: 1796438) at 37 +/- 1°C and 5% CO2.

Dose Groups
1. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
2. Positive Control: 4 µg/mL DNCB
3. Test Item: 8 concentrations of the test item (dose finding assay/ main experiment)

dose finding assay 1 and 2:
1000, 500, 250, 125, 62.50, 31.25, 15.63 and 7.81 µg/mL

main experiment 1 and 2:
60.63; 50.53; 42.10, 35.09; 29.24; 24.37; 20.30; 16.92 µg/mL

Pre-Experiments
Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB (CAS No.: 97-00-7, ≥99% purity,
Aldrich, Lot No.: BCBP8259V) at a final concentration of 4 µg/mL and nickel sulphate (CAS No.: 10101-97-0, ≥99% purity, Carl Roth, Lot No.: 384216612) at a final concentration of 100 µg/mL served as positive control while lactic acid (CAS No.: 50-21-5, ≥99% purity, Fluka, Lot No.: BCBQ0345V) at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

Solvent finding
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl (Braun; Lot No.: 160948002) at a final concentration of 100 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. Test items
not soluble in DMSO at 500 mg/mL, were solved at the highest soluble concentration in DMSO by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL. The test item was tested in the most applicable solvent at its highest concentration.

Experimental Procedure

Dose Finding Assay
Starting from 500 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally
used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution .

For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10E6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10E6 cells/well).

The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

After 24 h ± 1 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e.
FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer. 200 µL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL.

The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an exication wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells will be acquired and cell viability
will be calculated for each test concentration according to equation 10-1.

The CV75 value, i.e. the concentration showing 75% cell survival, will be calculated by log-linear interpolation utilizing equation 10-2. The CV75 value will be used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression
The test item was solved using DMSO as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2;
CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.

The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions will be further diluted 250-fold into the culture medium (working
solutions). These working solutions are finally used for cell treatment with a further final 2-fold dilution factor. Alternative concentrations were used upon justification (e.g. in case of poor solubility or cytotoxicity)

For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well). The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for for 24 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

After 24 h ± 1 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained
with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).

The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm or FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated according to equation 10-3. The cell viability was calculated according to equation 10-1.

Data Analysis
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 and Effective Concentration 200 values were performed using the software Microsoft Excel 2010. The
mean values and standard deviations of the single replicates were determined using the respective excel commands.

Calculation of Cell Viability 10.1

Cell Viability = Number of living cells x 100
Total number of aquired Cells

Calculation of CV75 - 10-2

Log CV75 = (75-c)*Log b-(75 -a)* Log d
a-c
a = the lowest cell viability over the threshold of 75% cell viability
b = the concentration of the test item, which causes a
c = the highest cell viability under the threshold of 75% cell viability
d = the concentration of the test item, which causes c

Calculation of the Realtive Fluoescence Intensity (RFI) - 10-3

RFI =MFI test item treated cells - MFI test item treated isotype cell
MFI solvent treated calls - MFI solvent treated isotype cells

RFI = relative fluorescence intensity
MFI = geometric mean fluorescence intensity

Evaluation of the Results

Prediction Model
For CD86/CD54 expression measurement, each test item was tested in at least two independent
runs to derive a single prediction. Each independent run was performed on a different day or on the
same day provided that for each run: independent fresh stock solutions and working solutions of the
test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86
and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was
equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two
independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a
cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54
were equal to or are greater than 150% and 200% respectively at any tested dose at a cell
viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should
be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x
CV75 is <90%. In contrast, a positive test outcome will be accepted irrespective of cell viabilities
>90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be
accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9%
NaCl solution; 1000 µg/mL for DMSO) even if the cell viability is >90%.

Effective Concentration 150 and Effective Concentration 200 values:
For test chemicals classified as sensitiser the effective concentration 150 for CD86 (EC150) and the effective concentration 200 for CD54 (EC200) can be calculated with following formula 10-4:

EC150 =Bdose +[( 150-BRFI)/(ARFI-BRFI)* (Adose -Bdose)]
EC200 =Bdose +[( 200-BRFI)/(ARFI-BRFI)* (Adose -Bdose)]

If the RFI value of the lowest dose is above the positive criteria of CD86 and CD54, EC150 and EC300 values can be calculated using the lowest dose by log linear extrapolation according to the following formula 10-5:

EC150 =2{log2(Bdose)-(150-BRFI)/(ARFI-BRFI)*[log2 (Adose -Bdose)]}
EC200 =2{log2(Bdose)-(200-BRFI)/(ARFI-BRFI)*[log2 (Adose -Bdose)]}

Adose is the lowest concentration in µg/mL with RFI > 150 (CD86) or 200 (CD54)
Bdose is the highest concentration in µg/mL with RFI < 150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54)


For the purpose of more precisely deriving the EC150 and EC200 values, three independent runs should be performed for CD86/CD54 expression measurement. The EC150 and EC200 values are the median value of the ECs calculated from three independent runs. In order to select the median
value, three independent runs are necessary. If only two of three independent runs meet the positive criteria, the higher EC150 or EC200 of the two calculated values is adopted. The EC values could potentially contribute to the assessment of sensitising potency [8] when used in integrated
approaches such as IATA [4].

Acceptance criteria:
The test meets acceptance criteria if:
- the cell viability of the solvent controls is >90%,
- the cell viability of at least four tested doses of the test item in each run is >50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

Key result

Run / experiment:

other: Experiment 1 and 2

Parameter:

other: Upregulation of CD54 and or CD86 in at least two independent experiment runs

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Reactivity Check of the Cell Stock

Doubling time of the cells was monitored and found to be 42.7 h which is within the doubling time range specified by the manufacturer (35 - 50 h).

Table 1: Results of the Cell Batch Activation Test

Sample	Concentration [µg/mL]	CD86		CD54		Activated
		Cell viability [%]	RFI	Cell viability [%]	RFI	yes/no
DNCB	4	85.8	393	85.6	408	Yes
NiSO4	100	90.4	272	90.4	332	Yes
LA	1000	98.1	56	98.5	56	No

 

The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and

CD86. The negative control LA did not induce an upregulation of CD54 and CD86.

The cell batch was accepted for further testing.

Solvent Finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in

DMSO (Applichem, Lot. No.: 0000803731) at a concentration of 500 mg/mL.

Dose Finding Assay

The dose finding assay was performed using stock solutions with a concentration of 500 mg/mL.

Table 2: Results of the Dose Finding Assay

Sample	Experiment 1		Experiment 2
	Concentration [µg/mL]	Cell viability [%]	Concentration [µg/mL]	Cell viability [%]
Medium Control	0	98.00	0	97.20
DMSO Control	-	97.80	-	96.70
Benzenesulfonic acid, mono- C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3- propanediamin	7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00	97.80 97.70 96.60 66.90 2.70 2.70 28.10 60.10	7.81 15.63 31.25 62.50 125.00 250.00 500.00 1000.00	97.00 97.10 96.00 64.10 1.40 1.10 60.70 23.90
Calculated CV75 [µg/mL]	51.73		49.32
Mean CV75 [µg/mL]	50.53
SD CV 75 [µg/mL]	1.71

 

 The mean CV75 was derived from two single runs and was found to be 50.53 ± 1.71 µg/mL. Based on the mean CV75, the main experiment was performed covering a concentration range from 60.63 – 16.92 µg/mL (30.32 – 8.46 mg/mL stock solutio

Results CD54 and CD86 Expression

For determination of the cell surface markers CD54 and CD86 two independent experiments were performed using separate cultivated cells at passage 19 (first experiment) and 21 (second experiment). For each experiment separately weighted samples and preparations were used.

Table 3: CD54 and CD86 Expression Experiment 1

 

Sample	Conc [µmL]	Cell Viability [%]			Mean Fluorescence Intensity			Corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	98.1	98.2	98.0	1545	939	805	740	134	87	77	192	117
DMSO Control	0.20%	98.1	98.2	98.1	1640	964	791	849	173	100	100	207	122
DNCB	4.00	88.7	87.9	87.2	4678	2440	889	3789	1551	446	897	526	274
Benzenesulfoic acid, mono C10- 13-alkyl derivs., compds. with N,N-dimethyl-1, 3- propanediamine	60.63	36.7	37.1	35.2	2328	1277	901	1427	376	168	217	258	142
	50.53	67.5	66.5	66.8	2526	1131	883	1643	248	194	143	286	128
	42.10	80.2	81.4	82.3	2290	1122	850	1440	272	170	157	269	132
	35.09	92.2	92.1	92.1	2024	1056	834	1190	222	140	128	243	127
	29.24	94.8	94.7	94.2	1872	1026	767	1105	259	130	150	244	134
	24.37	96.4	95.8	96.2	1753	984	767	986	217	116	125	229	128
	20.30	97.4	97.2	97.2	1796	1018	823	973	195	115	113	218	124
	16.92	97.3	97.3	97.4	1871	1076	862	1009	214	119	124	217	125

 

Table 4: CD54 and CD86 Expression Experiment 2

Sample	Conc [µmL]	Cell Viability [%]			Mean Fluorescence Intensity			Corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.8	97.3	97.1	1399	990	848	551	142	80	78	165	117
DMSO Control	0.20%	96.7	96.9	96.3	1534	1027	845	689	182	100	100	182	122
DNCB	4.00	82.2	82.6	81.1	3445	1799	864	2581	935	375	514	399	208
Benzenesulfoic acid, mono C10- 13-alkyl derivs., compds. with N,N-dimethyl-1, 3- propanediamine	60.63	47.9	46.7	46.0	2392	1316	1043	1349	273	196	150	229	126
	50.53	65.9	67.6	64.0	2189	1251	994	1195	257	173	141	220	126
	42.10	85.5	85.7	84.8	1935	1171	868	1067	303	155	166	223	135
	35.09	88.9	89.3	90.1	1814	1189	851	963	338	140	186	213	140
	29.24	95.1	94.8	94.8	1567	1138	879	688	259	100	142	178	129
	24.37	93.6	93.5	93.3	1805	1195	904	901	291	131	160	200	132
	20.30	96.4	96.6	96.4	1618	1156	857	761	299	110	164	189	135
	16.92	96.3	96.4	96.2	1684	1112	873	811	239	118	131	193	127

 

Table 5: Acceptance Criteria

Acceptance Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
Cell viability solvent controls [%]	>90	98.0 - 98.2	pass	96.3 – 97.3	pass
number of test dosed with viability >50% CD86	>4	7	pass	7	pass
number of test dosed with viability >50% CD54	>4	7	pass	7	pass
number of test dosed with viability >50% IgG1	>4	7	pass	7	pass
RFI of positive control of CD86	>150	446	pass	375	pass
RFI of positive control of CD54	>200	897	pass	514	pass
RFI of solvent control of CD86	<150	115	pass	125	pass
RFI of solvent control of CD54	<200	129	pass	128	pass
MFI ratio IgG1/CD86 for medium control [%]	>105	192	pass	165	pass
MFI ratio IgG1/CD86 for DMSO control [%]	>105	207	pass	182	pass
MFI ratio IgG1/CD54 for medium control [%]	>105	117	pass	117	pass
MFI ratio IgG1/CD54 for DMSO control [%]	>105	122	pass	122	pass

 

Table 6: Historical Data

Criterion	Mean	SD	N
Cell viability solvent controls [%]	96.1	2.8	130
number of test dosed with viability >50%	-	-	382
RFI of positive control of CD86	322.7	118.0	21
RFI of positive control of CD54	575.8	255.3	21
RFI of solvent control of CD86	114.6	15.8	20
RFI of solvent control of CD54	121.2	13.6	20
MFI ratio IgG1/CD86 for medium control [%]	230.2	67.7	22
MFI ratio IgG1/CD86 for DMSO control [%]	141.1	12.1	22
MFI ratio IgG1/CD54 for medium control [%]	254.6	82.7	22

 

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser in accordance with UN GHS category 1.

The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

There are currently three in-chemico / In-vitro skin sensitisation test methods specified in ECHA guidance, the other two tests for this substance, the DPRA and the Keratinosens were negative. Based on this one approach that has been proposed is to use a two or more out of three positive responses to trigger skin sensitisation classification, in which case Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamine could be considered not to require classification as a skin sensitiser. However the current ECHA guidance does not endorse this approach. Also the current ECHA guidance requires for substances classified as skin sensitisers that we classify as 1A or 1B, in-vitro results can only result in classification as category 1A. Based on this equivocal result with only one positive result in the h-CLAT, the available information is considered not to be sufficient for classification. Therefore an animal test is necessary to confirm if this substance should be classified as a skin sensitiser and if so which category.

Executive summary:

Summary Results

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

In the present study Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N,N-dimethyl-1,3-propanediamine was dissolved in DMSO. For the dose finding assay stock solutions with a concentration of 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 50.53 ± 1.71 µg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps:

60.63; 50.53; 42.10, 35.09; 29.24; 24.37; 20.30; 16.92 µg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Slight cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 36.7% (CD86), 37.1% (CD54) and 35.2% (isotype IgG1 control) in the first experiment and to 47.9% (CD86), 46.7% (CD54) and 46.0% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 194% in experiment 1 and 196% in experiment 2. The upregulation above the threshold of 150% was observed in both experiments at concentrations of 42.10 µg/mL (170% experiment 1, 155% experiment 2), 50.53 µg/mL (194% experiment 1, 173% experiment 2) and 60.63 µg/mL (168% experiment 1, 196% experiment 2).

The expression of the cell surface marker CD54 was upregulated to 217% in experiment 1. The upregulation above the threshold of 200% was observed at a concentration of 60.63 µg/mL.

Since the expression of cell surface marker CD86 clearly exceeded the threshold in two independent experiments, and cell surface marker CD54 clearly exceeded the threshold in one experiment the test item is considered to be a skin sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (446% experiment 1; 375% experiment 2) and 200% for CD54 (897% experiment 1; 514% experiment 2) were clearly exceeded.

Conclusion

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DPRA

Solutions of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) analyzed by the validated method in Lysine containing synthetic peptides appeared to show the test item to be a strong skin sensitizer. This was considered to be a false positive due to the presence of an extra peak in the chromatogram eluting at approximately 13 minutes, when the peak areas of the Lysine peptide peak and this extra peak were summed, the total peak area matched that of the positive controls in the Lysine assay. It was considered that this could be the result of ion-pairing interactions with the HPLC column used in the validated method. An investigation confirmed the false positive result was due to ion-pairing effects between the test item and the HPLC column used in the validated method. As the result generated when using the Lysine containing synthetic peptides was affected by these ion-pairing interactions, the Lysine result has been excluded and only the Cysteine result is used for classification.

The Cysteine result places Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16015) in the reactivity class of “no or minimal reactivity” and therefore it is predicted to be a non skin sensitizer.

Keratinosens

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

h-CLAT

Since the expression of cell surface marker CD86 clearly exceeded the threshold in two independent experiments, and cell surface marker CD54 clearly exceeded the threshold in one experiment the test item is considered to be a skin sensitiser.

Guinea Pig Maximisation test

In the treated group (treatment dose of 4%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.

In the control group (associated with the treatment dose of 4%), no macroscopiccutaneous intolerance reactions were recorded after the challenge phase.

In the treated group (treatment dose of 2%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.

In the control group (associated with the treatment dose of 2%), no macroscopiccutaneous intolerance reactions were recorded after the challenge phase.

In conclusion, in view of these results, under these experimental conditions, the test item Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamine does not have to be classified in category 1 as a skin sensitizer, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

No signal word or hazard statement is required. 

So the overall conclusion is that Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine does not require classification as a skins sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

There are no validated animal tests for respiratory sensitisation. Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine does not require classification as a skins sensitiser therefore it is not expected to be a respiratory sensitiser.

Justification for classification or non-classification

The in-vitro testing of Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine produced negative results in the DPRA and Keratinosens assays. It was however positive in the h-CLAT assay. The current ECHA Guidance does not provide any overall prediction model as to how to combine the data from the three main non-animal test methods as detailed for this substance.  Various prediction models have been proposed but the proposal of (Bauch et. al. 2012) where there is a requirement for at least two out of the three tests to be positive for a test substance to be considered a skin sensitiser has gained some acceptance as a pragmatic approach to removing false positives responses based on a single positive response. Recently this approach has been found, for a group of 9 difficult substances, which are false positive in the local lymph node assay, (Kreiling et.al.2017) to provide a much lower rate of false positive results. The use of the two out of three prediction model reduced the number of false positive results and identified correctly the true positive substance tested and four true negatives. usual to get a single positive result, which does not mean the tests substance is automatically considered to be a skin sensitiser. Based on the results using the 2 out of three prediction model Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine would not be classified as a skin sensitisers. However in the absence of clear QSAR prediction concerning the potential for skin sensitisation, the ECHA guidance indicates that an animal study is required to clarify the skin sensitisation potential for classification and labelling. The default animal study is the local lymph node assay, however the OECD guideline 429 for the LLNA accepts that for some irritant substances such as surfactants this can give false positive results. For these the Guinea pig maximisation test is the alternative following guideline OECD406.

 

The Guinea pig maximisation test did not produce any indication of skin sensitisation at either the 4% or 2% challenge concentrations. Based on these results, under these experimental conditions, the test item Benzenesulfonic acid, C10-13-alkyl derivs., compds. with N,N-dimethyl-1, 3-propanediamine does not have to be classified in category 1 as a skin sensitizer, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

No signal word or hazard statement is required. So the overall conclusion is that Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine does not require classification as a skins sensitiser.

There are no validated animal tests for respiratory sensitisation. Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine does not require classification as a skin sensitiser this together to no known cases of respiratory sensitisation in workers confirms that . Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine does not require classification as a respiratory sensitiser.

References

ReinhardKreiling, Helge Gehrke, Thomas H. Broschard, Birte Dreeßen, Dorothea Eigler, David Hart, Veronika Höpflinger, Marcus Kleber, Joanna Kupny, Qiang Li, Peter Ungeheuer and, Ursula G. Sauer. In chemico, in vitro and in vivo comparison of the skin sensitizing potential of eight unsaturated and one saturated lipid compounds. Regulatory Toxicology and Pharmacology 90(2017) 262-276.

ECHA (2017)European Chemicals Agency Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.7a: Endpoint specific guidance, version 6.0: ECHA-17-G-18-EN, July 2017

Bauch, C., Kolle, S.N., Ramirez, T., Fabian, E., Mehling, A., Teubner, W., van Ravenzwaay, B.,Landsiedel, R., Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regulatory. Toxicology and. Pharmacology(2012). 63, 489-504.