(3E)-3-[[4-(2-carboxyethylcarbamoyl)phenyl]hydrazinylidene]-6-oxocyclohexa-1,4-diene-1-carboxylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th June 2018 - 19th July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”.
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 an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP

Test material

Test material information

Constituent 1

Reference substance name:

(3E)-3-[[4-(2-carboxyethylcarbamoyl)phenyl]hydrazinylidene]-6-oxocyclohexa-1,4-diene-1-carboxylic acid

EC Number:

617-116-8

Cas Number:

80573-04-2

Molecular formula:

C17 H15 N3 O6

IUPAC Name:

(3E)-3-[[4-(2-carboxyethylcarbamoyl)phenyl]hydrazinylidene]-6-oxocyclohexa-1,4-diene-1-carboxylic acid

Test material form:

solid

Specific details on test material used for the study:

Batch No.: 0400
Purity: 96%
Appearance: Orange solid
Storage conditions: Room temperature

Results and discussion

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.83%.

In vitro / in chemico

Results

Key result

Run / experiment:

other: Test item

Parameter:

other: Mean peptide depletion (%)

Value:

2.41

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Reference controls

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes: The mean depletion of both peptides was 64.83%.

Any other information on results incl. tables

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. The test item was soluble in dist. water : acetonitrile 1:1 (v/v). No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using dist. water : acetonitrile 1:1 (v/v). All test item solutions were freshly prepared immediately prior to use

Precipitation and Phase Separation

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant

Co-elution with the Peptide Peaks

No relevant co-elution of the test item with any of the peptide peaks was observed

Depletion of the cysteine peptide depletion:

Cysteine peptide
Sample	Peak area at 220 nm	Peptide Conc. [mM]	Peptide depletion [%]	Mean peptide depletion [%]	SD of peptide depletion [%]	CV of peptide depletion [%]
Positive control	4.7290 4.7400 4.7210	0.1451 0.1454 0.1449	71.19 71.12 71.24	71.18	0.06	0.08
Test item	16.1620 16.0220 15.7820	0.4951 0.4908 0.4835	2.31 3.15 4.60	3.35	1.16	34.65

Depletion of the Lysine peptide

Cysteine peptide
Sample	Peak area at 220 nm	Peptide Conc. [mM]	Peptide depletion [%]	Mean peptide depletion [%]	SD of peptide depletion [%]	CV of peptide depletion [%]
Positive control	6.2940 5.9870 5.6530	0.2177 0.2071 0.1955	56.28 58.41 60.73	58.47	2.23	3.81
Test item	14.2650 14.3900 14.3550	0.4938 0.4981 0.4969	1.95 1.10 1.34	1.46	0.44	30.32

Categorization of the test item:

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

Prediction model	Prediction model 1 (Cysteine peptide and lysine peptide/ratio 1:10 and 1:50)			Prediction model 2 (cysteine peptide/test item ratio: 1: 10)
Test substance	Mean peptide depletion [%]	Reactivity category	Prediction	Mean peptide depletion [%]	Reactivity category	Prediction
Test item	2.41	Minimal reactivity	No sensitiser	3.35	Minimal reactivity	No sensitiser
Positive control	64.83	High reactivity	Sensitiser	71.18	Moderate reactivity	sensitiser

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given conditions of the study, the test item showed minimal reactivity towards both peptides. Therefore, the test item, balsalazide acid, might be considered as a non-sensitiser.