Registration Dossier

Administrative data

Description of key information

Conclusion of the skin sensitising potential of Bromoform based on results of a series of skin sensitisation tests.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
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.
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


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

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
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.  

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Phase: 09 July 2018 to 02 October 2018. Report Issue: 04 December 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 442E; In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation.
Version / remarks:
Further details of OECD 442E method: Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), October 2017
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assaywas performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. QA statement)
Type of study:
other: Dendritic Cell Activation
Justification for non-LLNA method:
The h-CLAT assay was conducted as part of a stepwise approach to the assessment of skin sensitisation of the test substance, as required by REACH from 11 October 2016.
Details on the study design:
TEST ITEM CONCENTRATIONS
78, 93, 112, 135, 161, 194, 233 and 279 µg/mL in the first run
161, 193, 232, 278, 334, 401, 481 and 577 µg/mL in the second run
112, 134, 161, 193, 232, 278, 334 and 401 µg/mL in the third run

The test item concentrations for use in the main test were determined from two range-finder XTT cytotoxicity tests.

CONTROLS
Positive Control: DNCB (2,4-dinitrochlorobenzene, CAS No: 97-00-7, Purity ≥ 99%)) final concentration: 2 and 3 µg/mL
Medium control: Culture Medium

THP-1 CELL CULTURE
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of the testing laboratory to allow the repeated use of the same cell culture batch in experiments. Thawed stock cultures were propagated at 37 °C in plastic flasks. The cells are sub-cultured twice weekly. The THP-1 cell suspension is incubated at 37 °C and 5 % CO2 atmosphere. Cells are used up to two months after thawing but the maximum passage number is 30.

The passage numbers of the used THP-1 cells were 11 and 10 in the XTT assays and 22, 24 and 25 in the h CLAT for runs 1, 2 and 3, respectively.

CULTURE MEDIUM
RPMI 1640 Medium, GlutaMAX™ Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) is used to culture the cells during the assay.

PREPARATION AND SEEDING OF THP-1 CELLS
On the day of the cytotoxicity experiment (XTT) a volume of 100 µL with a cell density of 0.9 -1 x 10E6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate prior to treatment.

For the main experiment (h-CLAT) 0.9 - 1 x 10E6 cells/well in a volume of 500 µL were seeded in a 24-well plate before the treatment.

TREATMENT OF THE CELLS
For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75 (The estimated concentration showing 75% cell viability). Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.

Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 245 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.

STAINING OF THE CELLS
At the end of the incubation period each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for staining. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1. All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.

SAMPLE PREPARATION FOR MEASUREMENT
After staining with the antibodies, the cells were washed twice (at 2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

FLOW CYTOMETRY
The expression of cell surface antigens (CD54, CD86) was analysed using a flow cytometry (FACSCalibur, Becton Dickinson GmbH) and Cellquest Pro 6.0 software. Before using the flow cytometer the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.


Positive control results:
The Relative Fluorescence Intensity (RFI) values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%, except for the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the second h-CLAT run exceeded the positive criteria.
Key result
Run / experiment:
other: Run 1 CD54 Antibody
Parameter:
other: % Relative fluorescence intensity (RFI)
Value:
200
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: This result was considered invalid because the highest test item concentration did not show cell vilability < 90%.
Key result
Run / experiment:
other: Run 1 CD86 Antibody
Parameter:
other: % Relative fluorescence intensity (RFI)
Value:
150
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: This result was considered invalid because the highest test item concentration did not show cell vilability < 90%.
Key result
Run / experiment:
other: Run 2 CD54 Antibody
Parameter:
other: % Relative fluoresence (RFI)
Value:
200
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: Run 2 CD86 Antibody
Parameter:
other: % Relative fluoresence (RFI)
Value:
150
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Run 3 CD54 Antibody
Parameter:
other: % Relative fluoresence (RFI)
Value:
200
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: Run 3 CD86 Antibody
Parameter:
other: % Relative fluoresence (RFI)
Value:
150
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

XTT Cytotoxicity Test Results (dose range-finding tests) - Determination of the Estimated Concentration Showing 75% Cell Viability (CV75)

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 156.3 µg/mL up to the highest tested concentration (5000 µg/mL) in the first XTT test and starting with the concentration of 625 µg/mL up to the highest tested concentration (5000 µg/mL) in the second XTT test (threshold of cytotoxicity: < 75%). Precipitations were observed in the four highest test item concentrations. However, the CV75 of the second XTT test was calculated independent of the observed precipitations. The mean CV75 value of both XTT tests was calculated as 232.2 µg/mL.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
This result will not be interpreted as a stand alone result but incorporated in a battery of test results to provide an overall conclusion as to the skin senstising status of the test substance.
Conclusions:
The test item Bromoform (EC number 200-854-6) activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

Introduction

The purpose of the study was to assess the potential for the test item to activate dendritic cells using the in vitro Human Cell Line Activation Test (h-CLAT). Activation of dendritic cells has been identified as a third key event in the Adverse Outcome Pathway for skin sensitisation. The study was designed to meet the requirements of the following guideline:

  • OECD Guidelines for the Testing of Chemicals: OECD 442E;In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation.Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), October 2017.

 

 

Method

The test items as a suspension/dispersion in culture medium was administered to THP-1 cells for 24 ± 0.5 hours in three independent runs. The highest test item concentration for the main experiment was previously determined by two XTT tests.The following concentrations of the test item were tested in the main experiments (h-CLAT):

78, 93, 112, 135, 161, 194, 233 and 279 µg/mL in the first run.

161, 193, 232, 278, 334, 401, 481 and 577 µg/mL in the second run.

112, 134, 161, 193, 232, 278, 334 and 401 µg/mL in the third run.

 

Culture medium, positive control (2,4-dinitrochlorobenzene) and solvent control (dimethyl sulfoxide) for the positive control were also run.

 

Results

The relative fluorescence intensity (RFI) of CD86 and CD54 expression was not equal to or greater than 150% and 200%, respectively at any dose in the first h‑CLAT run. However, this run with a negative result was not valid since the highest tested test item concentration did not show a cell viability < 90%. Therefore, the test item concentrations were adjusted for the second and third h‑CLAT run. The four highest tested test item concentrations of the second and the two highest tested test item concentrations of the third run were excluded from the evaluation, due to observed precipitations. The RFI of CD86 was equal to or greater than 150% in at least one concentration of both independent h-CLAT runs. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.

The positive control met the acceptance criteria for the test demonstrating the assay was functioning as expected.

Conclusion

The test item Bromoform (EC number 200-854-6) activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Phase: 09 July 2018 to 26 July 2018. Report Issued: 12 October 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
yes
Remarks:
There was a typographical error in the study plan concerning cell dilutions. The correct cell dilution was made on the study and hence this deviation is considered not to have had an effect on the reliability or scientific integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The KeratinoSens™ cell viability assay was conducted as part of a stepwise approach to the assessment of skin sensitisation of the test substance as required by REACH from 11 October 2016.
Details on the study design:
EXPERIMENTAL DESIGN

Test System
The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Duebendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM), supplemented with 50 mL foetal bovine serum and 5.5 mL Geneticin.

Replicates
Two independent experiments were performed.

Preparation of the test Item
A stock solution of the test item was prepared by weighing between 20 – 40 mg into a tared glass container and diluting in dimethyl sulfoxide (DMSO) to 200 mM, which is the highest concentration recommended by the test guideline.

Preparation of Test cell Cultures
The cells from flasks of actively growing cultures were detached and disaggregated and resuspended in fresh culture medium. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 µL volumes pipetted into all wells except well H12 of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. Well H12 of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

Luciferase Activity Measurment
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega. After incubation the test medium was removed from the wells by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Cel Viability Measurement
A kit (Molecular Probes Vybrant MTT kit V13154) was used to determine cell viability. The cell culture medium was removed from the wells of the 96-well plate and 100 µL fresh assay medium was added to each well along with 10 µL of MTT solution. The plate was incubated at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.
Positive control results:
The positive control, cinnamic aldehyde, passed the acceptance criteria in both experiments.
Key result
Run / experiment:
other: Run 1
Parameter:
other: Luciferase activity induction (Imax)
Value:
1.86
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Run 2
Parameter:
other: Luciferase activity induction (Imax)
Value:
1.23
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Run 1
Parameter:
other: Cellular viability (%)
Value:
90.08
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: An IC30 or IC50 could not be calculated.
Key result
Run / experiment:
other: Run 2
Parameter:
other: Cellular viability (%)
Value:
92.08
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: An IC30 or IC50 could not be calculated.

The Imax for the test substance was 1.86 in test 1 and 1.23 in test 2. The Imax for test 1 was >1.5 fold and statistically significant as compared to the DMSO control. However, as the luciferase induction >1.5 fold was only observed at one intermediate concentration, 15.63 µM, and was not dose dependent, this was not considered indicative of a positive response. 

The cellular viability did not fall below 90.08% in test 1 and 92.08% in test 2 and therefore the IC30 and IC50 could not be calculated. The EC1.5 for the test substance was 9.02 µM in test 1 and could not be calculated for test 2. No dose-response for luciferase induction was observed. 

The EC1.5 values of the positive control, cinnamic aldehyde were 10.18 μM and 16.09 μM for test 1 and 2, respectively, which lay within the historical control range for the laboratory.

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 11.9% and 14.8% for test 1 and 2, respectively, which met the acceptance criterion of below 20% for the test.

Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that Bromoform (EC 200-854-6) gave a negative response for skin sensitisation in the ARE-Nrf2 Luciferase Test (KeratinoSens™).
Executive summary:

Introduction

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, Bromoform (EC 200-854-6), is a skin sensitiser using the ARE-Nrf2 Luciferase Test (KeratinoSens™). The method used was designed to meet the requirements of OECD guideline 442D.

Method

Cells used in the assay were transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct cultured in 96-well flat-bottomed microtitre plates. Cells were treated with the test substance at 12 concentrations ranging from 0.98 and 2000 µM for 24 hours at 37 °C in a humidified atmosphere of 5% CO2 in air. Positive and solvent controls were run simultaneously. At the end of the incubation period cell viability was measured using the MTT assay and luciferase activity was measured using a Steady Glo® Luciferase Assay system kit. Two independent experiments were performed.

Results

The Imax for the test substance was 1.86 in test 1 and 1.23 in test 2. The Imax for test 1 was >1.5 fold and statistically significant as compared to the DMSO control. However, as the luciferase induction >1.5 fold was only observed at one intermediate concentration, 15.63 µM, and was not dose dependent, this was not considered indicative of a positive response.

Cellular viability did not fall below 90.08% in test 1 and 92.08% in test 2 and therefore the IC30and IC50could not be calculated. The EC1.5for the test substance was 9.02 µM in test 1 and could not be calculated for test 2. Graphs for luciferase induction showed no overall dose-response.

The validation and acceptance criteria for the test overall and the positive and negative solvent controls were met.

Conclusion

It was concluded that Bromoform (EC 200-854-6) gave a negative response for skin sensitisation in the ARE-Nrf2 Luciferase Test (KeratinoSens™).

 

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
Report Isssued 01 March 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
According to Annex VII (Regulation (EC) No 1907/2006) and the testing strategies for the 2018 registration deadline (ECHA Guidance, Chapter R.7a, 2016), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step.

Before starting any new testing an assessment of all available in vitro data, in vivo data, historical human data, data from valid (Q)SARs and data from structurally related substances (read-across approach) is required.
Qualifier:
no guideline followed
Principles of method if other than guideline:
As no relevant existing data skin skin sensitisation potential was found following searches a QSAR assessment was undertaken to meet the requirements of conducting skin sensitisation assessment for the test substance according to Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006 and Commission Regulation (EU) 2016/1688).

The QSAR assessment is the first step in a stepwise approach to assessing the skin sensitising potential of a substance by consideration of the structural properties of the test substance and any available existing data.
GLP compliance:
no
Justification for non-LLNA method:
QSAR assessment is recognised as a key first step in a battery of assessments of a test substance for skin sensitisation potential for registration under REACH. Depending upon the outcome further assessment may, or may not be required.
Key result
Parameter:
other: QSAR and expert assessment of skin sensitisation potential.
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: Some QSAR evidence of skin sensitising potential. Follow up confirmatory testing required as specified by Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006 and Commission Regulation (EU) 2016/1688).

The prediction of the skin sensitisation potential of Bromoform was performed with QSAR models TOPKAT V4.5, CAESAR (with VEGA 1.1.4 extension), Derek V5.0.2 and Toxtree V2.6.13. The assessment with OECD QSAR Toolbox V4.1 was based on the profiler and read-across prediction.

 

TOPKAT predicted Bromoform to be sensitiser; however, prediction was low reliable due to low structural similarity of structural analogues, moderate concordance of analogues with the predicted value and moderate accuracy.

 

CAESAR predicted Bromoform to be a non-sensitiser with low reliability (ADI: 0.446) as the query structure was indicated that it is outside applicability domain of the model.

 

DEREK did not match Bromoform for any alert. The reasoning level of the prediction was equivocal.

 

Toxtree identified an alert for SN2 Nucleophilic Aliphatic Substitution in respect to skin sensitisation reactivity domains for Bromoform.

OECD QSAR Toolbox gave no alerts for protein binding or for metabolites / transformation products.

The weight of evidence of the assessed models suggests a poor data set covering the structural analogues to Bromoform. The TOPKAT and CAESAR (VEGA) predictions are low reliable and not consistent. Only Toxtree indicates the SN2 nucleophilic aliphatic substitution for Bromoform, whereas all other tools trigger no alerts related to Bromoform skin sensitisation protential. No found or not matched alerts may be also a reason for the poor dataset.

 

The Toxtree alert follows a mechanistic pathway where bromine of bromoform is acting as a good leaving group. In addition, by analogy to chloroform, it cannot be excluded that Bromoform will covalently bind to the proteins.

 

Taking into account the prediction results on skin sensitising, toxicokinetic, physico-chemical properties of Bromoform as well as read-across assumptions there is an evidence that Bromoform may be a skin sensitiser. To confirm the assessed result and to cover the remaining key events in the cellular level, further in chemico and in vitro testing of Bromoform using OECD test methods 442C, 442D and 442E has to be performed.

Interpretation of results:
study cannot be used for classification
Remarks:
The expert assessment was not conclusive as to whether the test substance could be skin sensitiser and recommended further investigations.
Conclusions:
Taking into account the prediction results on skin sensitising, toxicokinetic, physico-chemical properties of Bromoform as well as read-across assumptions there is an evidence that Bromoform may be a skin sensitiser; however, for confirmation it is proposed that the in chemico/in vitro tests OECD 442C, 442D and 442E are conducted on Bromoform. If the outcome of these tests is inconclusive or positive and the data do not permit classification and risk assessment, then conduct of an OECD 429 LLNA is considered justified.
Executive summary:

Introduction

An assessment of the skin sensitisation potential of Bromoform was conducted based on a review of existing data, QSAR and expert assessment to addresses the requirements of conducting skin sensitisation assessment according to Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006 and Commission Regulation (EU) 2016/1688).

 

The assessment(s) form the first part of stepwise approach to assessing skin sensitisation potential. Depending on the results further assessments and / or in vivo in vitro testing may be required.

 

Method

Assessment of existing data was undertaken by screening the following databases: ChemIDplus, Chemspider, SRC's Fate Pointer File, ECHA, EFSA and TOXLINE.  The prediction of the skin sensitisation potential was performed with QSAR models TOPKAT V4.5, CAESAR V2.1.6 (with VEGA 1.1.4 extension), Derek V5.0.2 and Toxtree V2.6.13. Assessment with OECD QSAR Toolbox V4.1 was based on the profiler and read-across prediction.

 

Results

The databases examined gave no information on the skin sensitising properties of Bromoform.

The weight of evidence of the models used suggested a poor data set covering structural analogues to Bromoform and hence predictions are considered to have low reliability.

TOPKAT predicted Bromoform to be sensitiser while Toxtree indicated an alert for SN2 nucleophilic aliphatic substitution which may indicate skin sensitising potential. None of the other tools indicated alerts for skin sensitisation.

 

Conclusion

Taking into account the prediction results on skin sensitising, toxicokinetic, physico-chemical properties of Bromoform as well as read-across assumptions there is an evidence that Bromoform may be a skin sensitiser; however, for confirmation it is proposed that the in chemico / in vitro tests OECD 442C, 442D and 442E are conducted on Bromoform.

If the outcome of these tests is inconclusive or positive and the data do not permit classification and risk assessment, then conduct of an OECD 429 LLNA is considered justified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Bromform was tested in a series of in vitor skin senstisation assays followign completion of a QSAR assessment which indicated that Bromoform had some properties alerting for skin sensitisation, although the over a conclusion was considerd to be of low relability. Bromform was negative for skin sensitisation in the Direct Peptide assay and KeratinoSens ™ assay; however, Bromoform gave a positive response in the h-CLAT assay. As the testing procedure is a stepwise process, assessment of skin sensitisation potential is based on consideration of all test results and data.

Evidence from an OECD 431 in vitro skin corrosion test indicates that Bromoform is corrosive to skin and therefore a follow up confirmatory in vivo OECD 429 LLNA is not considered justified, as severe irritant or corrosive effects on the skin is likely to prevent or confound interpretation. 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Bromoform gave negative results in an in vitro KeratinoSens assay and a direct peptide reactivity assay. It was; however, positive in an h-CLAT assay whichquantifies changes in the expression of cell surface markers associated with the process of activation of monocytes and dendritic cells. 

 

In vitro testing of Bromoform for skin sensitisation was initiated following an initial QSAR assessment where two models used (TOPKAT and Toptree) indicated alerts for skin sensitisation with Bromoform, while two other (DEREK and CAESAR) predicted it negative. However, because of the poor dataset covering structural analogues these predictions were considered of low reliability and therefore only indicators that further testing should be conducted.

 

It is widely considered that skin sensitisation requires a number of steps referred to as an Adverse Outcome Pathway (AOP)1.  If one or more of these steps does not occur this is taken to indicate that a substance will not have the potential to cause skin sensitisation. Given that Bromoform is negative for skin sensitisation based in two out of three in vitro tests measuring different steps in the AOP, it has been concluded that Bromoform does not have the potential to cause skin sensitisation.  

Bromoform has been used as an industrial chemical (mainly as an intermediate) for many years and there is no indication in the scientifc literature (TOXNET database) that Bromoform can or has caused skin sensitisation.

Reference

1OECD (2014), The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins, OECD Series on Testing and Assessment, No. 168, OECD Publishing, Paris. (https://doi.org/10.1787/9789264221444-en)