Bromoform

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase:09 July 2018 to 12 July 2018. Report Issue: 04 October 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:

The direct peptide binding assay is a recognised assay for conducting as part of a battery of tests for the assessment of skin sensitisation for registration under REACH.

Test material

Test material information

Constituent 1

Reference substance name:

Bromoform

EC Number:

200-854-6

EC Name:

Bromoform

Cas Number:

75-25-2

Molecular formula:

CHBr3

IUPAC Name:

tribromomethane

Test material form:

liquid

In vitro test system

Details on the study design:

Experimental Procedure

Assessment of Test Item Solubility: The solubility of Bromoform was assessed in acetonitrile.

Preparation of Peptide Stock Solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards: Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference Controls and Precision Controls: Stability controls) and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solution: The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of Bromoform was prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls: Accurate volume aliquots of Bromoform and the positive control were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of Bromoform and 5 mM of the positive control. For the co-elution control, acetonitrile was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls: Accurate volume aliquots of Bromoform and the positive control were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of Bromoform and 25 mM of the positive control. For the co-elution control, acetonitrile was used in place of the Lysine stock solution.

Incubation: The appearance of the test substance and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis: The concentration of both the Cysteine and Lysine peptides in the presence of the test substance and the associated positive controls was quantified by HPLC using UV detection.

Instrument: Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm
Column Temerature:30°C
Sample temperature: 25°C
Detector wavelength: UV, 220 nm

Results and discussion

Positive control results:

The positive control substance, Cinnamic Aldehyde, responded as a expected demonstrating that the assay was working as expected.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cystiene

Parameter:

other: Mean peptide depletion by Bromoform (%)

Value:

-0.579

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Lysine

Parameter:

other: Mean peptide depletion by Bromoform (%)

Value:

0.065

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The solubility of Bromoform in acetonitrile at a nominal concentration of 100 mM was achieved.

Any other information on results incl. tables

The depletion of peptide in the presence of test substance was as follows:

	Mean peak area of reference control (µV.sec)	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by the test substance (%)
Cysteine	Stability Control : 747360 (n=6)	751690 (n=3)	-0.579
Lysine	Stability Control: 818700 (n=6)	818170 (n=3)	0.0650

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Solutions of Bromoform were successfully analysed using a validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. With “no or minimal” depletion of both peptides, the test substance is predicted by DPRA as negative and to not be a potential skin sensitiser based on this assay.

Executive summary:

Introduction

The capability of Bromoform to react with proteins was assessed using a method designed to be be compatible with the following method: OECD 442C: In Chemico Skin Sensitisation Direct Peptide Reactivity Assay (DPRA). The rationale of the assay is that if a chemical is capable of reacting with proteins then it has the potential to act as a sensitiser.

Method

The percentage depletion over time of two synthetic peptides (containing respectively a cysteine and a lysine amino acid) from peptide mixtures following an approximate 24 hour (22-26 hours) incubation with the test item was measured. The percentage of peptide depletion was calculated by High Performance Liquid Chromatography using ultra-violet detection.

Results

The depletion of peptide in the presence of test substance was as follows:

	Mean peak area of reference control (µV.sec)	Mean peak area of peptide with test item (µV.sec)	Mean peptide depletion by the test substance (%)
Cysteine	Stability Control: 747360 (n=6)	751690 (n=3)	-0.579
Ltsine	Stability Control: 818700 (n=6)	818170 (n=3)	0.0650

Conclusion

Solutions of Bromoform were successfully analysed using a validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. With “no or minimal” depletion of both peptides, the test substance is predicted by DPRA as negative and to not be a potential skin sensitiser based on this assay.