Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

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
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

Constituent 1
Chemical structure
Reference substance name:
Hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2-undecanone
EC Number:
270-151-7
EC Name:
Hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2-undecanone
Cas Number:
68411-85-8
Molecular formula:
C19H32N4O2
IUPAC Name:
2-{[(6-oxocyclohexa-2,4-dien-1-ylidene)methyl]amino}guanidine; undecan-2-one
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 the 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
Run / experiment:
other: mean of three replicates
Parameter:
other: CV75 (ug/mL)
Remarks:
estimated concentration causing 75% viability
Value:
56.2
Run / experiment:
other: CD54 Main Assay #1
Parameter:
other: EC200 (ug/mL)
Value:
32.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Run / experiment:
other: CD86 Main Assay #1
Parameter:
other: EC150 (ug/mL)
Value:
67.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Run / experiment:
other: CD54 Main Assay #2
Parameter:
other: EC200 (ug/mL)
Value:
32.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Run / experiment:
other: CD86 Main Assay #2
Parameter:
other: EC150 (ug/mL)
Value:
67.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not valid
Key result
Run / experiment:
other: CD54 Main Assay #3
Parameter:
other: EC200 (ug/mL)
Remarks:
concentration showing an RFI value >200 in CD54
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
Run / experiment:
other: CD86 Main Assay #3
Parameter:
other: EC150 (ug/mL)
Remarks:
concentration showing an RFI value >150 in CD86
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
Run / experiment:
other: CD54 Main Assay #4
Parameter:
other: EC200 (ug/mL)
Remarks:
concentration showing an RFI value >200 in CD54
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
Run / experiment:
other: CD86 Main Assay #4
Parameter:
other: EC150 (ug/mL)
Remarks:
concentration showing an RFI value >150 in CD86
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
Run / experiment:
other: CD54 Main Assay #5
Parameter:
other: EC200 (ug/mL)
Remarks:
concentration showing an RFI value >200 in CD54
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
Run / experiment:
other: CD86 Main Assay #5
Parameter:
other: EC150 (ug/mL)
Remarks:
concentration showing an RFI value >150 in CD86
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).