Benzoic acid, 2-([1,1'-biphenyl]-4-ylcarbonyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Precipitates of the test substance were observed at the end of the incubation with the peptides, therefore the peptide depletion may be underestimated.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

2-{[1,1'-biphenyl]-4-carbonyl}benzoic acid

EC Number:

700-862-4

Cas Number:

42797-18-2

Molecular formula:

C20 H14 O3

IUPAC Name:

2-{[1,1'-biphenyl]-4-carbonyl}benzoic acid

Test material form:

solid: particulate/powder

In chemico test system

Details on the study design:

Reactivity (expressed as percent of peptide depletion) is determined following 24-h contact between the test substance and peptide in acetonitrile at the ratios 1:10 cysteine: test substance and 1:50 lysine: test substance by liquid chromatography with UV-Visible spectra detection.

The reactivity of the test substance was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test substance and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test substance for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

% depletion = 1- (Peptide peak area in replicate injection/Mean peptide peak area in relevant reference control C samples) x 100

The test substance was dissolved at 100 mM in a 1:9 mixture of DMSO : acetonitrile. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test substance did not co-elute with either the lysine or the cysteine peptides.

Analytical analysis:
- High Performance Liquid Chromatography (HPLC) Systems with a UV detector (220 nm),
- analytical chromatographic columns (Zorbax SB C18, 100 x 2.1 mm; 3.5 μm HPLC), in-line filter C18, 4.0 x 2.0 mm (Phenomenex)
Mobile phases run in gradient:
A: acetonitrile + 0.085 % TFA
B: milli-Q water + 0.1% TFA
Flow: 350 μL/minute
Oven temperature: 30.0 °C
Autosampler temperature: nominal temperature: +25 °C
Injection volume: 5 μL
Total analysis time: 20 minutes

Co-elution control samples preparation:
For the co-elution control with cysteine peptide: 50 μL of test substance formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide: in parallel, 250 μL of test substance formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

Reference control samples preparation:
Reference control A and B samples: In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
Reference control C samples:
Reference control C samples were prepared for each solvent used to dissolve the test and positive control substances.
For the reference control C prepared with cysteine peptide:
50 μL of each vehicle (a 1:9 mixture of DMSO:acetonitrile or acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reference control C prepared with lysine peptide:
In parallel, 250 μL of each vehicle (a 1:9 mixture of DMSO:acetonitrile or acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

Cinnamaldehyde (positive control) depletion control samples preparation:
For the reactivity of cinnamaldehyde with cysteine peptide:
50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide:
In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Test substance samples preparation:
For the reactivity of test substance with cysteine peptide:
50 μL of test substance formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of substance with lysine peptide:
In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

Preparation of the calibration curve samples:
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20 % acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.


Skin sensitisation (In chemico test system) - Details on study design:
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Results and discussion

Positive control results:

The positive control was cinnamaldehyde (purity: 98.9%).
As several test items were assayed concurrently, the results of the positive control were shared.
The positive control was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in acetonitrile at 100 mM. The physical aspect of the formulation was clear colorless solution. The formulation was used just after its preparation.

In vitro / in chemico

Results

Key result

Parameter:

other: mean of the percent cysteine and percent lysine depletion

Value:

0.97

Vehicle controls validity:

valid

Remarks:

1:9 mixture of DMSO:acetonitrile

Negative controls validity:

valid

Remarks:

co-elution control samples

Positive controls validity:

valid

Remarks:

cinnamaldehyde

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

DATA ANALYSIS

Calculation of the percent peptide depletion
Each appropriate peak was integrated and the peak area for calibration standards, control and test substance samples were determined. Based on the concentration of standards and their peak area, a linear calibration curve was generated. Then, the concentration of peptide was determined in each sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of peptide using the linear calibration curves. Then, for each positive control and test substance replicate, the percent depletion of peptide was determined from the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent) by using the following formula:

% depletion = 1- (Peptide peak area in replicate injection/Mean peptide peak area in relevant reference control C samples) x 100

Then, the mean percent depletion of the three replicates was calculated for each peptide as well as the mean of the percent cysteine and percent lysine depletions. Negative depletion values were considered as "Zero" for the calculation of the mean %depletion.

Evaluation of the possible co-elution of the test substance with the lysine or cysteine peptides:
In order to detect possible co-elution of the test substance with a peptide, chromatograms of the co-elution control samples were analyzed and compared with those of the reference control C samples.

ACCEPTANCE CRITERIA

The run was considered valid if the following criteria were fully met:
. the calibration curve should have a coefficient of determination (r2) ≥ 0.99,
. the mean peptide concentrations of the reference control A samples should be within ± 10 % of the nominal concentration,
. the cinnamaldehyde depletion control samples should meet the following acceptance criteria:
− for the cysteine peptide, the mean percent depletion value should be between 60.8 and 100 % with a SD < 14.9%,
− for the lysine peptide, the mean percent depletion value should be between 40.2 and 69.0 % with a SD < 11.6%,
. the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile must be < 15.0%.
The test substance results were considered valid if the following criteria were fully met:
. the mean peptide concentrations of the reference control C samples prepared in the appropriate solvent should be within ± 10% of the nominal concentration,
. the maximum SD for the test substance replicates should be < 14.9% for the percent cysteine depletion value and < 11.6% for the percent lysine depletion value.

Any other information on results incl. tables

The cysteine peptide depletion value was 1.21%,

The lysine peptide depletion value was 0.72%.

The mean of the percent cysteine and percent lysine depletions was equal to 0.97%. However, since precipitates were observed at the end of the incubation with the peptides, the peptide depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test substance was considered to have minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative and the test substance is likely not to have any potential to cause skin sensitization, though with limitations due to test substance precipitation with the peptides.

Applicant's summary and conclusion

Interpretation of results:

other: Negative

Conclusions:

Under the study conditions, the test substance was considered to have no/minimal peptide reactivity, although with limitations due to test substance precipitation with the peptides. Therefore, the test substance is considered to produce negative results in the DPRA assay.

Executive summary:

A study was conducted to determine the reactivity potential of the test substance PBBA, with synthetic cysteine and lysine peptides according to OECD Guideline 442 C (direct peptide reactivity assay (DPRA)), in compliance with GLP. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test substance for 24 h at the ratios 1:10 cysteine: test substance and 1:50 lysine: test substance. The test substance was dissolved at 100 mM in a 1:9 mixture of DMSO : acetonitrile. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with UV detection at 220 nm.Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide area in the three relevant reference control C samples. Results of the test substance reactivity indicated for the cysteine peptide, the mean depletion value of 1.21% and for the lysine peptide, the mean depletion value of 0.72%. The mean of the percent cysteine and percent lysine depletions was equal to 0.97%. However, since precipitates were observed at the end of the incubation with the peptides, depletion may be underestimated. Since the mean of the percent cysteine and percent lysine depletions was <6.38%, the test substance was considered to have no/minimal peptide reactivity. The acceptance study criteria were satisfied, the study was therefore considered to be valid. Under the study conditions, the test substance was considered to have no/minimal peptide reactivity, although with limitations due to test substance precipitation with the peptides. Therefore, the test substance was considered to produce negative results in the DPRA assay (Chevallier, 2017).