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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Remarks:
Human Cell Line Activation Test
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01 Oct 2017 - 16 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 442E In-Vitro Skin Sensitization: Human Cell Line Activation Test (hCLAT)
Version / remarks:
29 July 2016
Deviations:
no
Principles of method if other than guideline:
- Principle of test: The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.
GLP compliance:
yes
Type of study:
activation of dendritic cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: BASF (Puebla, Mexico) batch# 6191104
- Expiration date of the lot/batch: August 2018
- Purity test date: 08 March 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature
- Stability under test conditions: stable under conditions of assay
- Solubility and stability of the test substance in the solvent/vehicle: soluble in vehicle based on initial solubility screen

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received

In vitro test system

Details on study design:
The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers

THP-1 cells are cultured, at 37°C under 5% CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 μg/mL streptomycin. The positive controls for this assay are 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity) and the negative control, is lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity).

A dose finding assay is performed to determine the CV75, being the test chemical concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value is used to determine the concentration of test chemicals for the main assay involving CD86/CD54 expression measurement. The test substance working solutions are mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well or 96-well flat-bottom plate. The treated plates are then incubated for 24±0.5 hours at 37°C under 5% CO2. After 24±0.5 hours of exposure, CD54/86 expression is measured by FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies at 4°C for 30 min. Cell viability was measured by Propidium Iodine staining.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Cell viabilities of medium and solvent/vehicle controls should be > 90%. The RFI values of both CD86 and CD54 should not exceed positive crtieria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%). The MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
-Acceptance criteria for positive control: 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%.
-Acceptance criteria for test chemicals: Cell viability should be more than 50% in at least four tested concentrations. Negative results are acceptable only for test chemicals exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5000 ug/mL is used as the maximal test concentration of a test chemical than a negative result is acceptable even if the cell viability is above 90%.

Results and discussion

Positive control results:
The positive control 2,4-dinitrochlorobenzene (DNCB) produced positive results (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in three out of five replicates. The positive control nickel sulfate (NiSO4) produced positive results (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in all five replicates.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: CV75 (ug/mL)
Remarks:
estimated concentration causing 75% viability
Run / experiment:
mean of three replicates
Value:
56.2
Parameter:
other: EC200 (ug/mL)
Run / experiment:
CD54 Main Assay #1
Value:
32.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Parameter:
other: EC150 (ug/mL)
Run / experiment:
CD86 Main Assay #1
Value:
> 67.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Parameter:
other: EC200 (ug/mL)
Run / experiment:
CD54 Main Assay #2
Value:
32.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Parameter:
other: EC150 (ug/mL)
Run / experiment:
CD86 Main Assay #2
Value:
> 67.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Key result
Parameter:
other: EC200 (ug/mL)
Remarks:
concentration showing an RFI value >200 in CD54
Run / experiment:
CD54 Main Assay #3
Value:
56.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC150 (ug/mL)
Remarks:
concentration showing an RFI value >150 in CD86
Run / experiment:
CD86 Main Assay #3
Value:
> 67.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: EC200 (ug/mL)
Remarks:
concentration showing an RFI value >200 in CD54
Run / experiment:
CD54 Main Assay #4
Value:
> 67.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: EC150 (ug/mL)
Remarks:
concentration showing an RFI value >150 in CD86
Run / experiment:
CD86 Main Assay #4
Value:
> 67.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: EC200 (ug/mL)
Remarks:
concentration showing an RFI value >200 in CD54
Run / experiment:
CD54 Main Assay #5
Value:
27.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC150 (ug/mL)
Remarks:
concentration showing an RFI value >150 in CD86
Run / experiment:
CD86 Main Assay #5
Value:
> 67.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The RFI values of both CD86 and CD54 did not exceed positive crtieria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) for the negative control Lactic Acid in any of five replicates.

-Acceptance criteria for positive control: The positive control 2,4-dinitrochlorobenzene (DNCB) did not meet the crtieria for acceptance (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in the first two replicates, tehrefore the results of the test substance were considered invalid. The assay was repeated with 3 additional replicates, and DNCB met the acceptance criteria for these additional replicates. The positive control nickel sulfate (NiSO4) met the acceptance crtieria in all 5 replicates.

Applicant's summary and conclusion

Interpretation of results:
other: Sensitizing
Conclusions:
The in-vitro Human Cell Line Activation Assay (hCLAT) was used to assess Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone for its potential to activate dendritic cells through measurement of markers CD86 and CD54. A positive result was obtained for CD54 (RFI≥ 200%) in two out of three replicates which resulted in an overall positive h-CLAT prediction. However, this study only gives information on one key event of the skin sensitization AOP and should be assessed with other information within a defined approach or weight of evidence.
Executive summary:

In an OECD TG 442E hCLAT assay, Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was exposed to THP-1 cells at 67.5, 56.2, 46.7, 38.8, 32.3, 27.0, 22.5, and 18.8 ug/ml based on the initial cytotoxicity screens. A positive result was obtained for CD54 (RFI≥ 200%) in two out of three replicates which resulted in an overall positive h-CLAT prediction. Based on these results, Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone would be predicted as capable of activating dendritic cells (key event #3). Therefore the results of this assay are considered positive and should be used within a weight of evidence for concluding on the skin sensitization potential of Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).