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EC number: - | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07-June-2021 to 01-July-2021 (experimental dates)
- Reliability:
- 1 (reliable without restriction)
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
- Version / remarks:
- June 2018
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- human Cell Line Activation Test (h-CLAT)
Test material
- Reference substance name:
- Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol
- Molecular formula:
- not applicable
- IUPAC Name:
- Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol
- Test material form:
- solid: particulate/powder
- Details on test material:
- Appearance: beige powder
Constituent 1
- Specific details on test material used for the study:
- Description: Light brown powder
Storage conditions: Stored at 15-25°C, protected from light.
In vitro test system
- Details of test system:
- THP-1 cell line [442E]
- Details on the study design:
- Preliminary solubility data indicated that BTMR was not directly soluble in culture medium (RPMI-1640) or saline. Solubility was however achieved in DMSO at a concentration of 110 mg/mL.
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The test article was formulated at 110 mg/mL in DMSO and then diluted a series of times with the corresponding solvent and in culture medium. The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 1.72 220 µg/mL. As there was no effect on viability in the dose finding assay and no CV75 was obtained, eight stock solutions of test article were prepared by 1.2-fold serial dilutions to give eight concentrations, and the stock solutions were then further diluted 250-fold into the culture medium (working solutions). The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate. Final test concentrations in a range from 61.4 to 220 µg/mL (after dilution in medium) were used for the expression measurements.
THP-1 cells were cultured in a humidified incubator set to 37ºC, 5% CO2, in RPMI 1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin. Cell density did not exceed 1 x 106 cells/mL, and cells did not exceed 30 passages.
Reactivity check was performed using DNCB, nickel sulphate and lactic acid two weeks after thawing. Only cells which passed the reactivity check were used for the assay.
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2). After the 24-hour incubation period, all cells from the 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL). PI uptake was analyzed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.
Cell viability and the CV75 value were calculated.
Two independent runs (experiments) were needed to drive a prediction. Each independent run was performed on a different day. On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium and distributed into a 24-well plate. The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes. After blocking, the cells were centrifuged and stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed three times with an excess of FACS buffer, resuspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).
The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry. - Vehicle / solvent control:
- DMSO
- Positive control:
- other: 2,4-dinitrochlorobenzene (DNCB)
Results and discussion
- Positive control results:
- RFI values were 150% for CD86 and 200% for CD54, and cell viability was >50% in each independent run.
In vitro / in chemico
Results
- Key result
- Group:
- test chemical
- Run / experiment:
- mean
- Parameter:
- EC150, CD86 [442E]
- Value:
- 140.49 µg/mL
- Outcome of the prediction model:
- positive [in vitro/in chemico]
- Other effects / acceptance of results:
- In Experiment 1, the RFI values for CD86 were >150% (with cell viability >50%) at multiple concentrations and the RFI values for CD54 were <200% at all concentrations. In Experiment 2, the RFI values for CD86 were >150% and the RFI values for CD54 were >200% at one or more concentrations (with cell viability >50%). The test article therefore gave a positive prediction in the assay.
The EC150 value for CD86 calculated by linear regression of endpoint assay data was 140.49 µg/mL. No EC200 value was calculated for CD54 as this marker was negative in Experiment 1.
All assay acceptance criteria were met.
- The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run.
- In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI =150% and CD54 RFI =200%).
- For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
- For the positive control, RFI values were =150% for CD86 and =200% for CD54, and cell viability was >50% in each independent run.
- For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.
Any other information on results incl. tables
Table 1 - BTMR relative fluorescence intensity (RFI)
Concentration (µg/mL) | RFI (CD86) | RFI (CD86) | RFI (CD54) | RFI (CD54) |
Exp 1 | Exp 2 | Exp 1 | Exp 2 | |
61.40 | 112 | 97 | 79 | 93 |
73.68 | 196 | 113 | 164 | 108 |
88.41 | 167 | 115 | 94 | 103 |
106.10 | 189 | 142 | 121 | 157 |
127.31 | 169 | 127 | 92 | 116 |
152.78 | 134 | 171 | 96 | 144 |
183.33 | 146 | 153 | 107 | 240 |
220.00 | 130 | 111 | 121 | 142 |
Solvent/vehicle control (DMSO) | 100 | 147 | 89 | 116 |
Positive control (DNCB) | 534 | 431 | 1070 | 879 |
Applicant's summary and conclusion
- Interpretation of results:
- study cannot be used for classification
- Remarks:
- The data can only be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitizers and non-sensitizers for the purpose of hazard classification and labelling.
- Conclusions:
- In a human Cell Line Activation Test (h-CLAT), performed according to OECD TG 442E, BTMR was considered to be positive.
- Executive summary:
A human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.
For the dose finding assay, the test article was dissolved in dimethyl sulfoxide (DMSO) at a maximum attained of concentration of 110 mg/mL giving a maximum test concentration of 220 µg/mL. No reduction in viability was observed.
For the expression measurements, test concentrations in a range from 61.4 to 220 μg/mL (after dilution in medium) were used. Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10E6 cells per well. After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.
In Experiment 1, the RFI values for CD86 were >150% (with cell viability >50%) at multiple concentrations and the RFI values for CD54 were <200% at all concentrations. In Experiment 2, the RFI values for CD86 were >150% and the RFI values for CD54 were >200% at one or more concentrations (with cell viability >50%). The test article therefore gave a positive prediction in the assay.
The EC150 value for CD86 was calculated to be 140.49 μg/mL.
All acceptance criteria of the h-CLAT assay parameters were met in each experiment.
BTMR was considered to be positive in the human Cell Line Activation Test.
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