4-Ethyl-2’-fluoro-4’-bromobiphenyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jun 03 -Sep 17, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Test material

Test material information

Constituent 1

Reference substance name:

4-bromo-4'-ethyl-2-fluoro-1,1'-biphenyl

EC Number:

812-029-3

Cas Number:

116713-40-7

Molecular formula:

C14H12BrF

IUPAC Name:

4-bromo-4'-ethyl-2-fluoro-1,1'-biphenyl

In chemico test system

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

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 65.69%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

0.82

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

0.69

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

Any other information on results incl. tables

Peptide Depletion in %

1) Cystein Depletion

Sample	Peak Area	Actual Peptide [mM]	Peak Are Ref Control C	Peptide Depletion [%]	Corr. peptide Depletion [%]
1	47.5989	0.475	47.8543	0.28	0.28
2	48.2216	0.482	48.4220	-1.02	0.00
3	46.6954	0.466	46.9235	2.17	2.17
Mean	47.5050	0.474	47.7333	0.48	0.82
Evaluation Reactivity Class. Negative

1) Lysine Depletion

Sample	Peak Area	Actual Peptide [mM]	Peak Are Ref Control C	Peptide Depletion [%]	Corr. peptide Depletion [%]
1	37.3825	0.477	37.3643	2.08	2.08
2	38.7725	0.496	38.4179	-1.56	0.00
3	40.5898	0.519	37.7442	-6.32	0.00
Mean	38.915	0.497	38.1755	-1.94	0.69
Evaluation Reactivity Class. Negative

Applicant's summary and conclusion

Interpretation of results:

other: The data generated with this test should be considered in the context of integrated approached such as IATA.

Conclusions:

The test item revealed a mean cysteine and lysine peptide depletion of 0.76% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Executive summary:

The purpose of this study was to determine the sensitising potential of the test item in a Direct Peptide Reactivity Assay (DPRA). The study was performed according to OECD guideline 442C. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

The test item was dissolved at a concentration of 100 mM in acetonitrile.

Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and acetonitrile were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate.

Test item-treated samples revealed a cysteine peptide depletion of 0.82% and a lysine peptide depletion of 0.69% (mean peptide depletion of 0.76%) and, hence, were well below 6.38%. The test item is considered negative and predicted to be a non sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.35% for cysteine and 65.40% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

The acceptance criteria of validity were fulfilled in this test.

The test item revealed a mean cysteine and lysine peptide depletion of 0.76% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).