4,4'-bis(chloromethyl)-1,1'-biphenyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 2017 to 31 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

1

Reference substance name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

EC Number:

216-784-4

EC Name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

Cas Number:

1667-10-3

Molecular formula:

C14H12Cl2

IUPAC Name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

Test material form:

solid: particulate/powder

Details on test material:

- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%), protected from humidity, under inert gas.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed and stored under appropriate conditions until ready to perform testing. It was dissolved in the selected vehicle (acetone) at 100 mM. This formulation had the aspect of a clear colorless solution. The formulation was used just after its preparation.

In chemico test system

Details on the study design:

VEHICLE
Several vehicles were tested during the solubility assay. The test item was found not soluble at 100 mM in acetonitrile, milli-Q water, 1:1 mixture of acetonitrile:milli-Q water and isopropanol, even after a sonication step of 1 minute. A solution was obtained at 100 mM in acetone (without sonication step). Therefore, this vehicle (acetone) was retained.
Based on solubility results, the retained vehicle was acetone.

POSITIVE CONTROL
The positive control was cinnamaldehyde. The positive control 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.

CO-ELUTION CONTROL SAMPLES
In order to detect possible co-elution of the test item with a peptide, co-elution control samples were prepared by incubating the test item formulation with each buffer used to dilute the peptides. Cysteine or lysine peptides were not added to these samples.

REFERENCE CONTROL SAMPLES
For each peptide, the analytical batch included reference control samples (sub-categorized in reference control A, B or C samples). All these control samples were prepared in triplicate and at the nominal concentration of 0.500 mM in the solvent. These samples were used to:
. Reference control A: Check the accuracy of the calibration curve for peptide quantification,
. Reference control B: Check the stability of the peptide during analysis,
. Reference control C: Check that the solvent did not impact the percentage of peptide depletion.

CYSTEINE PEPTIDE
. Peptide sequence: Ac-RFAACAA-COOH
. Peptide sequence synonyms: Ac RFAACAA-OH or RFAACAA-COOH
. Molecular weight: 750.88 g/mol
. Supplier: JPT Peptide Technologies GmbH
. Batch No.: 260515HSDWW0715
. Storage condition: At -20 °C
. Description: White powder

The cysteine peptide solution was freshly prepared at 0.667 mM in an aqueous phosphate buffer (pH 7.5) solution.

LYSINE PEPTIDE
. Peptide sequence: Ac-RFAAKAA-COOH
. Peptide sequence synonyms: Ac RFAAKAA-OH or RFAAKAA-COOH
. Molecular weight: 775.91 g/mol
. Supplier: JPT Peptide Technologies GmbH
. Batch No.: 220114HSDWW0715
. Storage condition: At -20 °C
. Description: White powder

The lysine peptide solution was freshly prepared at 0.667 mM in an aqueous ammonium acetate buffer (pH 10.2) solution.

EQUIPMENT, REAGENTS AND COMPUTER SYSTEMS
. 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 analytical column),


DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as follows.
- Preparation of the samples
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

- Co-elution control samples preparation
For the co-elution control with cysteine peptide: 50 μL of test item 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 item 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 items.

For the reference control C prepared with cysteine peptide: 50 μL of each vehicle (acetone 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 (acetone 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 item samples preparation
For the reactivity of test item with cysteine peptide: 50 μL of test item 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 test item 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).

- Incubation of the samples
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25 °C and protected from light before injection into the HPLC-UV system.
At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation.
Samples presenting precipitate were centrifuged at 400 g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

- 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.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

- HPLC/UV analysis of the samples
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
. one blank sample (peptide dilution buffer),
. one calibration curve injected at the beginning of the analytical batch,
. three reference control A samples,
. the co-elution control sample,
. three reference control B samples,
. reference control C sample (replicate 1),
. positive control sample (replicate 1),
. test item study sample (replicate 1),

The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3: Three reference control B samples.

HPLC CONDITIONS
The HPLC/UV method used for the samples analysis is summarized in the table below:
Analytical Column: Zorbax SB C18, 100 x 2.1 mm, 3.5 μm (Waters)
In-line filter C18, 4.0 x 2.0 mm (Phenomenex)
Mobile phase:
Mobile phase A: acetonitrile + 0.085 % TFA
Mobile phase B: milli-Q water + 0.1 % TFA
- Flow: 350 μL/minute
- Gradient:
Time % Mobile phase A % Mobile phase B
0 10 90
10 25 75
11 90 10
13 90 10
13.5 10 90
20 10 90

UV Wavelenght: 220 nm
Rinse solution: Acetonitrile
Oven temperature: 30.0 °C
Autosampler temperature: Nominal temperature of +25 °C
Injection volume: 5 μL
Retention times:
Cysteine-peptide: Approx. 9.8 minutes
Lysine-peptide: Approx. 7.5 minutes
Total analysis time: 20 minutes

DATA ANALYSIS AND CALCULATION
- Calculation of the percent peptide depletion
Each appropriate peak was integrated and the peak area for calibration standards, control and test item 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 item 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.
Peak areas and peptide concentrations are presented in the report. Standard Deviation (SD) and Coefficient of Variation (CV) were calculated and reported.

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

Results and discussion

In vitro / in chemico

Results

Key result

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Evaluation of the Presence of Precipitate at the end of the Incubation with Peptides

At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis.

As precipitate was observed in the test item, positive control and reference control samples incubated with the cysteine and lysine peptides as well as in co-elution samples prepared with the lysine or cysteine dilution buffer, these vials were centrifuged at 400 g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Thus, only supernatants were injected into the HPLC/UV system.

Evaluation of the Results

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 item did not co-elute with either the lysine or the cysteine peptides (% interference < 10 %). As a result, the mean percent depletion values were calculated for each peptide:

. for the cysteine peptide, the mean depletion value was 29.59 %,

. for the lysine peptide, the mean depletion value was 0.00 %.

The mean of the percent cysteine and percent lysine depletions was equal to 14.80 %. 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 between 6.38 and 22.62 %, the test item was considered to have a low peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization, though with limitations due to test item precipitation with the peptides.

Table 1. Determination of Cysteine Peptide and Lysine Peptide Depletion in Samples Spiked with a Solution at 100 mM of Test Material.

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) of test material	Depletion classification
	Peak area (μV/sec)	% depletion	Peak area (μV/sec)	% depletion
1 2 3	1228848 1201290 1158729	27.68 29.30 31.80	1551039 1553036 1552155	0.00* 0.00* 0.00*
Mean SD % CV	- - -	29.59 2.08 7.0	- - -	0.00* nc nc	14.80	Low reactivity
Precipitate:	Yes		Yes
Micelle	No		No

*: Value set to 0 due to negative depletion

-: not applicable

Table 2. Acetonitrile Determination of Cysteine Peptide and Lysine Peptide Depletion in Samples Spiked With a Solution at 100 mM of Cinnamaldehyde.

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) of Cinnamaldehyde	Depletion classification
	Peak area (μV/sec)	% depletion	Peak area (μV/sec)	% depletion
1 2 3	493840 514914 513144	72.95 71.79 71.89	625969 602903 65378	57.51 59.07 58.23
Mean SD % CV	- - -	72.21 0.64 0.9	- - -	58.27 0.78 1.3	65.24	High reactivity

Applicant's summary and conclusion

Interpretation of results:

other: Classification will be determined in conjuction with other sensitisation studies as part of an integrated testing strategy.

Conclusions:

Under the conditions of this study, the test item was considered to have a low peptide reactivity and may therefore have the potential to cause skin sensitisation, though with limitations due to test item precipitation with the peptides.

Executive summary:

The potential of the test material to act as a sensitiser was investigated in a GLP study in accordance with the standardised guidelines OECD 442C.

This test was conducted as part of a tiered strategy for skin sensitization assessment. The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item 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).

The test item was dissolved at 100 mM in acetone.

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 item did not co-elute with either the lysine or the cysteine peptides (% interference < 10 %). As a result, the mean percent depletion values were calculated for each peptide using the formula described in Data analysis and calculation. For the cysteine peptide, the mean depletion value was 29.59 % and for the lysine peptide, the mean depletion value was 0.00 %.

The mean of the percent cysteine and percent lysine depletions was equal to 14.80 %. 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 between 6.38 and 22.62 %, the test item was considered to have a low peptide reactivity. Therefore, the DPRA prediction is considered as positive and the test item may have potential to cause skin sensitization, though with limitations due to test item precipitation with the peptides.

Under the experimental conditions of this study, the test item was considered to have a low peptide reactivity, though with limitations due to test item precipitation with the peptides. The test item is considered positive in the DPRA assay.