[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The weight of evidence approach taken is explained in the provided endpoint summary.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

other: Weight of evidence

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 March 2021 to 01 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The weight of evidence approach taken is explained in the provided endpoint summary.

Reason / purpose for cross-reference:

other: Weight of Evidence

Reason / purpose for cross-reference:

other: Weight of Evidence

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

On Day 2 animal 49F (Prelim 2) and animal 50F (Prelim 3) did not receive the second observation. The animals received their first observations around midday and were health checked the following morning at 0759 and neither showed signs of ill health.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Housing
The animals were housed in groups of up to five during acclimatisation and housed in pairs from Day 1 in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014).
Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Covance.
No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study.

Water
Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been periodically analysed for specific contaminants.
No contaminants were present in water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Diet
5LF2 EU Rodent Diet 14%, was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.
No contaminants were present in diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Environment
The animal rooms were designed to permit a minimum of 15 air changes per hour. The target temperature and humidity ranges were 19 to 25°C and 40 to 70% respectively. Daily recordings of maximum and minimum temperature and humidity were made.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.

Environmental Enrichment
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and nesting materials. The nesting materials were removed from the cages prior to dosing on Day 1.

Vehicle:

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

Concentration:

10%, 25%, 50%

No. of animals per dose:

4

Details on study design:

Preliminary Screening Tests
In the absence of toxicological information regarding irritation and/or systemic toxicity, an initial preliminary screening test was conducted with one animal.
The mouse was treated by daily application of 25 µL of the undiluted test article to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and scored using the following scale:
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.
The animal was killed by an intraperitoneal injection of an overdose of sodium pentobarbitone at the end of the observation period. Death was confirmed by cervical dislocation.
Due to the clinical signs and erythema noted in the initial preliminary test, two further preliminary screening tests were conducted as described above with one animal per preliminary test, treated by daily application of 25 μL of the test article at a concentration of 50% or 25% v/v in 80% v/v acetone in olive oil.

Main Study
Groups of four female mice were assigned to study according to the table given below. Doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected on the basis of the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation.
Group Number Group Description Concentration Number of Animals in Group
1 Vehicle control - 4
2 Test - low concentration 10% v/v 4
3 Test - intermediate concentration 25% v/v 4
4 Test - high concentration 50% v/v 4

Inlife Procedures
Test Article Administration
Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate test or control formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.

Treatment Regimen
The four groups of four female mice were subjected to application of the vehicle control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3.
On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection. After this treatment, the mice were returned to their cages.
Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exsanguination under a deep plane of inhalation anaesthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.

Clinical Signs
Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article (see Protocol Deviations, Section 9).

Routine Health Checks
All animals were examined at the beginning and end of the working day throughout the acclimatisation and study periods to ensure they were in good health (see Protocol Deviations, Section 9).

Body Weights
Mice were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of 3HTdR.

Terminal Procedures
Recovery of Lymph Nodes
Once death had been confirmed each mouse was placed on a bench lined with paper with its ventral aspect uppermost. A mid-line incision from the lower jaw to the sternum was made and the cervical structures were exposed by reflecting the skin. The auricular lymph nodes were located and removed using curved end forceps. Any connective tissue was removed from the capsule of the nodes. The auricular lymph nodes of all mice from each dose group were placed into a petri dish containing 5 mL phosphate buffered saline.

Preparation for Scintillation Count
The lymph nodes collected into each petri dish were cut open and disaggregated by squashing the fragments with a sharp blade. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible.
After 5 minutes the pooled liquor was filtered into a second conical tube by transferring the liquor into a 10 mL syringe and passing it through a stainless steel gauze containing a fabric filter, cut to size (Clarcor UK, Lockertex Filtration Products, Warrington, UK). Any visible sediment remaining prior to filtering was left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes. Following centrifugation, the supernatant was discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This was centrifuged at 200 g for 10 minutes. The supernatant was discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8°C (nominal 4°C).
On the following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added.

Scintillation Counting
Once prepared the scintillation vials were placed in the appropriate carrier racks. Two background vials were prepared, one containing ca 10 mL of scintillation fluid and the other containing 1 mL of 5% w/v aqueous trichloroacetic acid and ca 10 mL scintillation fluid. The carrier rack was passed into the scintillation counter. All vials, including the background samples, were submitted for liquid scintillation counting for 10 minutes, using a 3H quench curve.
Incorporation of 3HTdR is measured by ß-scintillation counting as disintegrations per minute (DPM) over a ten-minute period. This value was corrected to account for the background containing 5% w/v aqueous trichloroacetic acid and scintillation fluid.

Positive control substance(s):

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

Positive control results:

The sensitivity and reliability of the test system has been checked using α hexylcinnamaldehyde (CAS Number 101 86 0). A Stimulation Index of 3.0 or greater is expected from this substance. The historical positive control data given in the Annex confirms adequate performance of the assay.

Key result

Parameter:

SI

Value:

5.03

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

5.5

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

4.77

Test group / Remarks:

10%

Cellular proliferation data / Observations:

Mortality
All animals survived treatment with Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine.

Clinical Signs
There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 10, 25 or 50% v/v formulations of the test article.
Greasy fur behind the ears and to the back of the neck were observed in all animals group 2 to 4 animals from Day 1 to Day 6. Slight redness behind the ears was also observed in Group 3 and 4 animals (Day 2 to Day 6).

Body Weights
There was no indication of a treatment related effect on body weight.

Stimulation Indices
Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls.
The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

Interpretation of results:

other: The substance has the potential to cause skin sensitisation.

Conclusions:

The Local Lymph Node Assay demonstrated that Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine has the potential to cause skin sensitisation.

Executive summary:

This study was conducted to assess the potential of the test article, Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine, to cause skin sensitisation in the mouse.

Following three preliminary screening tests using the undiluted test article, a 50% formulation and a 25% formulation, the main study was conducted using 50, 25 and 10% v/v formulations in 80% v/v acetone in olive oil.

Groups of four female CBA / CaCrl mice were subjected to topical applications of vehicle control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated ³H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from all animals in each group were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

	Concentration of Test Article in Applied Formulation (% v/v)
	10%	25%	50%
Stimulation Index	4.77	5.50	5.03

 

The Local Lymph Node Assay demonstrated that Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine does have the potential to cause skin sensitisation.

Cross-reference 2

Reason / purpose for cross-reference:

other: Weight of evidence

Reference

Endpoint:

skin sensitisation, other

Remarks:

QSAR

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Skin Sensitisation Isopropylamine

1. SOFTWARE
Nexus v.2.5.2 (Build 5, Jul 2022), Derek Nexus v6.2.1,


2. MODEL (incl. version number)
Derek KB 2022 2.0, skin sensitisation


3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Name: Isopropylamine
CAS No.: 75-31-0
SMILES: NC(C)C


4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: TOX 7.4.1 Skin sensitisation
- Unambiguous algorithm: Structural alerts, logic of argumentation, feature-based database search, nearest neighbours (within same alert as query compound) based on Tanimoto similarity
- Defined domain of applicability: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule, then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
- Appropriate measures of goodness-of-fit and robustness and predictivity: Not applicable.
- Mechanistic interpretation: Each alert includes a description of the proposed mechanism.


5. APPLICABILITY DOMAIN
- Prediction: skin sensitisation in mammal is NON-SENSITISER – interpreted as negative.
- Applicability domain: Query structure is within the applicability domain because no misclassified or unclassified features were reported.
- Analogues: The query structure does not match any structural alerts or examples for skin sensitisation in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser.
- mechanism: Not applicable


6. ADEQUACY OF THE RESULT
The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin.



Skin Sensitisation Constituent 3
1. SOFTWARE
Nexus v.2.5.2 (Build 5, Jul 2022), Derek Nexus v6.2.1,


2. MODEL (incl. version number)
Derek KB 2022 2.0, skin sensitisation


3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Name: Octyl Benzene sulphonic acid (branched, secondary connectivity), compound with isopropylamine (1:1) (OBS)
CAS No.: -
SMILES: C1=C(C=CC(=C1)S(=O)(=O)[O-])C(CC(CCC)C)C.[NH3+]C(C)C


4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: TOX 7.4.1 Skin sensitisation
- Unambiguous algorithm: Structural alerts, logic of argumentation, feature-based database search, nearest neighbours (within same alert as query compound) based on Tanimoto similarity
- Defined domain of applicability: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule, then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
- Appropriate measures of goodness-of-fit and robustness and predictivity: Not applicable.
- Mechanistic interpretation: Each alert includes a description of the proposed mechanism.


5. APPLICABILITY DOMAIN
- Prediction: skin sensitisation in mammal is NON-SENSITISER – interpreted as negative.
- Applicability domain: Query structure is within the applicability domain because no misclassified or unclassified features were reported.
- Analogues: The query structure does not match any structural alerts or examples for skin sensitisation in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser.
- mechanism: Not applicable


6. ADEQUACY OF THE RESULT
The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin.



Skin Sensitisation Constituent 10
1. SOFTWARE
Nexus v.2.5.2 (Build 5, Jul 2022), Derek Nexus v6.2.1,


2. MODEL (incl. version number)
Derek KB 2022 2.0, skin sensitisation


3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Name: Pentadecyl Benzene sulphonic acid (branched, secondary connectivity), compound with isopropylamine (1:1) (PBS)
CAS No.: -
SMILES: C1=C(C=CC(=C1)S(=O)(=O)[O-])C(CC(CC(CC(CC(C)C)C)C)C)C.[NH3+]C(C)C


4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: TOX 7.4.1 Skin sensitisation
- Unambiguous algorithm: Structural alerts, logic of argumentation, feature-based database search, nearest neighbours (within same alert as query compound) based on Tanimoto similarity
- Defined domain of applicability: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule, then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
- Appropriate measures of goodness-of-fit and robustness and predictivity: Not applicable.
- Mechanistic interpretation: Each alert includes a description of the proposed mechanism.


5. APPLICABILITY DOMAIN
- Prediction: skin sensitisation in mammal is NON-SENSITISER – interpreted as negative.
- Applicability domain: Query structure is within the applicability domain because no misclassified or unclassified features were reported.
- Analogues: The query structure does not match any structural alerts or examples for skin sensitisation in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser.
- mechanism: Not applicable


6. ADEQUACY OF THE RESULT
The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin.


This endpoint study record is part of a Weight of Evidence approach comprising two studies, namely OECD 442E and OECD 429 detailed in section 7.4.1, as well as a QSAR prediction for skin sensitisation (this study).

This data is sufficient to fulfil the information requirements and determine if the substance is likely to be a strong skin sensitiser in humans, as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

other: Weight of Evidence

Reason / purpose for cross-reference:

other: Weight of Evidence

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on (Q)SAR

Principles of method if other than guideline:

- Software tool(s) used including version: Nexus v.2.5.2 (Build 5, Jul 2022), Derek Nexus v6.2.1
- Model(s) used: Derek KB 2022 2.0, skin sensitisation

GLP compliance:

not specified

Remarks:

Not applicable

Justification for non-LLNA method:

Not applicable for QSAR.

Key result

Remarks on result:

other: Not applicable for QSAR, refer to "Any other information on results incl. tables".

Results from (Q)SAR:

Substance queried: Isopropylamine

The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin. 

 

Substance queried: Octyl Benzene sulphonic acid (branched, secondary connectivity), compound with isopropylamine (1:1) (OBS):

The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin. 

 

Substance queried: Pentadecyl Benzene sulphonic acid (branched, secondary connectivity), compound with isopropylamine (1:1) (PBS):

The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin. 

Conclusions:

Results from (Q)SAR:

Substance queried: Isopropylamine

The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin. 


Substance queried: Octyl Benzene sulphonic acid (branched, secondary connectivity), compound with isopropylamine (1:1) (OBS):

The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin. 


Substance queried: Pentadecyl Benzene sulphonic acid (branched, secondary connectivity), compound with isopropylamine (1:1) (PBS):

The query structure does not activate an alert for skin sensitisation and no unclassified or misclassified features were reported. Thus, the prediction is considered reliable. The query structure is suggested to be a non-sensitising to skin. 

Executive summary:

The skin sensitisatin potential of three substances was estimated using the (Q)SAR Model Derek KB 2022 2.0, skin sensitisation. This is a valid model for which the substances fall under their applicability domain as explained in the "Justification for type of information", see attached QMRF. 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine

IUPAC Name:

Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine

Test material form:

liquid

Details on test material:

Molecular weight: 357.269
Appearance: light amber viscous liquid
Storage conditions: room temperature, in the dark

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

442E

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test article was prepared shortly before testing. Preparation was conducted under subdued lighting with the aid of vortex mixing, ultrasonication (for approximately 10 minutes) and warming at 37°C.
- Preparation of the test chemical serial dilutions: The test article was formulated at 500 mg/mL in DMSO then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent. The stock solutions were then further diluted 250-fold in culture medium (working solutions).
- Preparation of the positive controls: 2,4-dinitrochlorobenzene (DNCB) prepared in DMSO (2 mg/mL stock), diluted in culture medium to obtain a working solution of 8 µg/mL, and final treatment concentration of 4 µg/mL in the plate.
- Preparation of the solvent, vehicle and negative controls: The solvent/vehicle control was dimethyl sulphoxide (DMSO) diluted in culture medium for the test article solutions to obtain a final concentration of 0.2% in the treatment plate.
- Stable dispersion obtained: No data
- Log Kow of the test chemical: No data available
- Other:

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 1000 µg/mL
- Solubility in solvents: soluble in DMSO
- Solubility in incubation medium: reliminary solubility data indicated that Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine was not directly soluble in culture medium (RPMI-1640) or saline, but was soluble in dimethyl sulphoxide (DMSO) at approximately 500 mg/mL. Accordingly, DMSO was chosen as the vehicle for this assay. Subsequent dilution of the DMSO formulation in culture medium indicated that the solubility limit in culture medium was in excess of 1000 µg/mL, as indicated by a lack of any visible precipitation at this concentration 24 hour after test article addition.
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: mean CV75 29.22 µg/mL.
- Final concentration range selected on basis of: Eight stock solutions of each test article were prepared by 1.2-fold serial dilutions using the corresponding solvent, and then further diluted 250-fold into the culture medium to give eight working solutions ranging from 0.335 x CV75 to 1.2 x CV75. The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 9.79-35.06 µg/mL.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 1
- Number of repetitions: 1
- Test chemical concentrations: 9.79, 11.74, 14.09, 16.91, 20,29, 24.35, 29.22, 35.06 µg/mL
- Application procedure
- Exposure time: After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes.
After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes).
After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.
- Study evaluation and decision criteria used
- Description on study acceptance criteria: The cell viabilities of medium and solvent control should be higher than 90%
In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%)
For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%
In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%
For the test article, the cell viability should be more than 50% in at least four tested doses in each run.
- Other:

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): N/A
- Incubation conditions: The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37°C, 5% CO2).
- Washing conditions: The cells were washed at least
twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of Propidium iodide= 0.625 µg/mL).
- Other: N/A

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: Propidium iodide uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.
- Plate used: 24-well plate (500 µL/1 x 10^6 cells per well).
- Propidium iodide staining/cytotoxicity measurements
- Preparation for CD54 and/or CD86 expression measurements/cell staining
- Other: The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated


DATA EVALUATION
- Cytotoxicity assessment: A biphasic cytotoxicity profile was observed. For run 1, after an initial decrease in viability to 4.1% at 125 μg/mL, the cell viability recovered again to ~90% viability at
a dose of 500 μg/mL, and then subsequently decreased again to 74.4% at 1000 μg/mL.
For run 2 there was again an initial decrease in viability to ~ 11% at 125 μg/mL,
followed by an increase (this time to ~67% viability at 500 μg/mL) and then a
subsequent decline again. As the observation of a biphasic cytotoxicity profile was
replicated in both runs it was therefore considered to be a true response.
- Prediction model used: An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered NEGATIVE:
• The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%)
• The RFI of CD54 is ≥200% at any tested concentration (with cell viability ≥50%).

Vehicle / solvent control:

DMSO

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

In vitro / in chemico

Results

Key result

Parameter:

EC200, CD54 [442E]

Value:

15.08 µg/mL

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

DOSE FINDING ASSAY
A biphasic cytotoxicity profile was observed. For run 1, after an initial decrease in viability to 4.1% at 125 μg/mL, the cell viability recovered again to ~90% viability at a dose of 500 μg/mL, and then subsequently decreased again to 74.4% at 1000 μg/mL. For run 2 there was again an initial decrease in viability to ~ 11% at 125 μg/mL, followed by an increase (this time to ~67% viability at 500 μg/mL) and then a subsequent decline again. As the observation of a biphasic cytotoxicity profile was replicated in both runs it was therefore considered to be a true response. Two CV75 values were indicated for run 1; a lower CV75 value of 37.4 μg/mL, and an additional higher CV75 value of 974.81 μg/mL. The CV75 value for run 2 was 21.03 μg/mL as the second viability increase did not exceed 75% in this run. A mean CV75 of 29.22 μg/mL was therefore calculated for the Dose Finding Assay (based on the lower CV75 values identified in each run).


CD86/CD54 EXPRESSION RESULTS
In Experiment 1, the RFI values for CD86 were <150% and the RFI values for CD54 were >200% at concentrations of 16.91-35.06 μg/mL (with cell viability >50%). In Experiment 2, the RFI values for CD86 were <150% at all concentrations and the RFI values for CD54 were >200% at 9.79 μg/mL (with cell viability >50%). The test article therefore gave a positive prediction in the assay. The EC200 value for CD54 calculated by linear regression of endpoint assay data was 15.08 μg/mL. No EC150 value was calculated for CD86 as this marker was negative in both experiments.


ASSAY ACCEPTANCE CRITERIA RESULTS
All assay acceptance criteria were met.
The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run.
In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.
For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Any other information on results incl. tables

	RFI (CD86)		RFI (CD54)
Concentration (μg/mL)	Exp 1	Exp 2	Exp 1	Exp 2
9.79	81	73	172	227
11.74	102	70	172	153
14.09	92	75	178	145
16.91	94	52	241	102
20.29	90	74	291	133
24.35	95	82	322	195
29.22	103	89	259	145
25.06	129	107	325	198
Solvent/vehicle control (DMSO)	96	127	53	95
Positive control (DNCB)	478	463	3091	1416

Applicant's summary and conclusion

Interpretation of results:

other: Based on this study alone, data not sufficient for sub-categorisation in accordance with CLP.

Conclusions:

The test article, Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine, was considered to be positive in the human Cell Line Activation Test.

Executive summary:

The study was conducted to investigate the potential of Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.
For the dose finding assay, the test article was dissolved in dimethyl sulphoxide (DMSO) at a concentration of 500 mg/mL giving a maximum test concentration of 1000 µg/mL. A reduction in viability was noted in the range finding assay, resulting in a mean CV75 of 29.22 µg/mL.
For the expression measurements, test concentrations in a range from 9.79 to 35.06 μg/mL (after dilution in medium) were used.
Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well.
After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.
The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.
In both experiments, the RFI values for CD86 were <150% and the RFI values for CD54 were >200% at one or more concentrations (with cell viability >50%). The test article therefore gave a positive prediction in the assay.
The EC200 value for CD54 was calculated to be 15.08 μg/mL.
All acceptance criteria of the h-CLAT assay parameters were met in each experiment.
The test article, Benzenesulfonic acid, mono-C9-13-branched alkyl derivs., compds. with isopropylamine, was considered to be positive in the human Cell Line Activation Test.