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EC number: 208-634-1 | CAS number: 536-45-8
- 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:
- weight of evidence
- Study period:
- 28 Aug - 07 Nov 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 442E (Skin Sensitization: Human Cell Line Activation Test (h-CLAT))
- Version / remarks:
- adopted in 2016
- Deviations:
- yes
- Remarks:
- cytotoxicity measurement and estimation of CV75 value for the dose finding assay was performed by XTT test instead of flow cytometry
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- p-anisic acid
- EC Number:
- 202-818-5
- EC Name:
- p-anisic acid
- Cas Number:
- 100-09-4
- Molecular formula:
- C8H8O3
- IUPAC Name:
- 4-methoxybenzoic acid
Constituent 1
In vitro test system
- Details on the study design:
- TEST CELL LINE
- Source: American Type Culture Collection (ATCC)
- Passage number: 9 (both XTT assays); 22 and 8 (h-CLAT runs 1 and 2, respectively)
CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% fetal bovine serum, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin
CONTROLS
Negative control
- Substance: culture medium
Solvent control for positive control
- Substance: 0.2% dimethyl sulfoxide (DMSO)
Positive control
- Substance: 2 and 3 µg/mL 2,4-dinitrochlorobenzene (DNCB)
EXPOSURE CONDITIONS
- Exposure duration: 24 ± 0.5 h
TEST CONCENTRATIONS
- Justification for top dose: Cytotoxic effects were observed at 1250 µg/mL in the first XTT test and at 625 and 1250 µg/mL in the second XTT test (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 819.4 µg/mL.
- Test concentrations: 274, 329, 395, 474, 569, 683, 819 and 983 μg/mL
NUMBER OF REPLICATIONS: single measurement in two independent experiments
CYTOTOXICITY
- Method: Cytotoxicity was determined by two independent XTT tests with different cell cultures to obtain a reliable CV75; the mean of two CV75 values was used to determine the dose-range for the main experiment. At the end of the 24 h incubation period with different test item concentrations, 50 µL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wave length 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1). A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance (= viability in [%]) as compared to the solvent control was calculated.
MEASUREMENT
- Device: FACSCalibur, Becton Dickinson GmbH
- Software: Cellquest Pro 6.0
STAINING
- Antibodies: FITC-labelled anti-CD86, FITC-labelled anti-CD54 and FITC-labelled mouse IgG1
EVALUATION CRITERIA/ PREDICTION MODEL
- For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline): The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%); the RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%). Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).
ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
- cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control
- in the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%)
- for both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%
- in the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations
- for the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90% (OECD 442E guideline).
Results and discussion
- Positive control results:
- The positive control 2,4-dinitrochlorobenzene (DNCB) test at final concentrations of 2 and 3% led to upregulation of the cell surface markers CD54 and CD86.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: First run
- Parameter:
- other: relative fluorescence intensity of CD86 (%)
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- since the RFI of CD86 is > 150% the prediction for sensitisation is considered positive
- Key result
- Run / experiment:
- other: Second run
- Parameter:
- other: relative fluorescence intensity of CD86 (%)
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- since the RFI of CD86 is > 150% the prediction for sensitisation is considered positive
- Key result
- Run / experiment:
- other: First run
- Parameter:
- other: relative fluorescence intensity of CD54 (%)
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- since the RFI of CD54 is > 200% the prediction for sensitisation is considered positive
- Key result
- Run / experiment:
- other: Second run
- Parameter:
- other: relative fluorescence intensity of CD54 (%)
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- since the RFI of CD54 is > 200% the prediction for sensitisation is considered positive
- Other effects / acceptance of results:
- OTHER EFFECTS:
- No test item-induced cytotoxicity was noted during the two runs of the main experiment.
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The laboratory has demonstrated technical proficiency for the h-CLAT by successfully testing the 10 profiency chemicals outlined in the OECD 442E guideline.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for DMSO control: The RFI values of the negative control of both CD86 and CD54 were 100, respectively, and thus did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The cell viability was 100.0 % and thus > 90%.
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 were > 650 and > 230, respectively, and thus exceeded the positive criteria (CD86 > 150% and CD54 > 200%) and the cell viability was > 60 % and thus > 50%.
Any other information on results incl. tables
Table 1: Results first h-CLAT run
|
Concentration (µg/mL) |
Antibody / ISO |
MFI GeoMean(FITC) |
MFI- ISO |
RFI (%) |
Cyto(Geo) GeoMean(7-AAD) |
Mean Cyto |
Viability (%) |
Medium control |
- |
ISO |
2.62 |
|
|
2.85 |
2.7 |
100.0 |
CD54 |
3.26 |
0.64 |
100.0 |
2.83 |
||||
CD86 |
5.12 |
2.50 |
100.0 |
2.46 |
||||
DMSO control |
- |
ISO |
2.19 |
|
|
3.06 |
2.7 |
100.0 |
CD54 |
3.26 |
1.07 |
100.0 |
2.73 |
||||
CD86 |
5.01 |
2.82 |
100.0 |
2.42 |
||||
Positive control (DNCB) |
2 |
ISO |
2.92 |
|
|
4.82 |
3.7 |
74.9 |
CD54 |
5.44 |
2.52 |
235.5 |
4.07 |
||||
CD86 |
17.05 |
14.13 |
501.1 |
2.07 |
||||
3 |
ISO |
3.25 |
|
|
6.26 |
4.6 |
59.9 |
|
CD54 |
6.68 |
3.43 |
320.6 |
4.99 |
||||
CD86 |
22.22 |
18.97 |
672.7 |
2.45 |
||||
Test Item |
274 |
ISO |
2.41 |
|
|
3.22 |
2.9 |
93.1 |
CD54 |
3.14 |
0.73 |
114.1 |
2.93 |
||||
CD86 |
4.57 |
2.16 |
86.4 |
2.59 |
||||
329 |
ISO |
2.40 |
|
|
3.15 |
2.9 |
93.7 |
|
CD54 |
3.20 |
0.80 |
125.0 |
2.96 |
||||
CD86 |
4.98 |
2.58 |
103.2 |
2.58 |
||||
395 |
ISO |
2.43 |
|
|
3.13 |
2.9 |
94.7 |
|
CD54 |
3.29 |
0.86 |
134.4 |
2.93 |
||||
CD86 |
4.99 |
2.56 |
102.4 |
2.54 |
||||
474 |
ISO |
2.57 |
|
|
3.09 |
2.8 |
98.0 |
|
CD54 |
3.38 |
0.81 |
126.6 |
2.85 |
||||
CD86 |
5.32 |
2.75 |
110.0 |
2.37 |
||||
569 |
ISO |
2.59 |
|
|
3.07 |
2.8 |
96.9 |
|
CD54 |
3.53 |
0.94 |
146.9 |
2.88 |
||||
CD86 |
5.57 |
2.98 |
119.2 |
2.45 |
||||
683 |
ISO |
2.64 |
|
|
3.10 |
2.8 |
98.2 |
|
CD54 |
3.60 |
0.96 |
150.0 |
2.81 |
||||
CD86 |
5.76 |
3.12 |
124.8 |
2.38 |
||||
819 |
ISO |
2.88 |
|
|
3.36 |
2.9 |
94.5 |
|
CD54 |
4.17 |
1.29 |
201.6 |
3.01 |
||||
CD86 |
7.14 |
4.26 |
170.4 |
2.24 |
||||
983 |
ISO |
2.79 |
|
|
3.08 |
2.9 |
92.2 |
|
CD54 |
3.90 |
1.11 |
173.4 |
2.82 |
||||
CD86 |
6.84 |
4.05 |
162.0 |
2.93 |
Table 1: Results second h-CLAT run
|
Concentration (µg/mL) |
Antibody / ISO |
MFI GeoMean(FITC) |
MFI- ISO |
RFI (%) |
Cyto(Geo) GeoMean(7-AAD) |
Mean Cyto |
Viability (%) |
Medium control |
- |
ISO |
2.44 |
|
|
3.85 |
3.7 |
100.0 |
CD54 |
3.30 |
0.86 |
100.0 |
3.78 |
||||
CD86 |
5.05 |
2.61 |
100.0 |
3.61 |
||||
DMSO control |
- |
ISO |
2.57 |
|
|
4.15 |
3.7 |
100.0 |
CD54 |
3.40 |
0.83 |
100.0 |
3.71 |
||||
CD86 |
5.01 |
2.44 |
100.0 |
3.12 |
||||
Positive control (DNCB) |
2 |
ISO |
3.63 |
|
|
5.03 |
4.6 |
79.8 |
CD54 |
7.18 |
3.55 |
427.7 |
4.70 |
||||
CD86 |
26.79 |
23.16 |
949.2 |
4.03 |
||||
3 |
ISO |
3.92 |
|
|
6.12 |
5.7 |
63.9 |
|
CD54 |
10.08 |
6.16 |
742.2 |
5.66 |
||||
CD86 |
19.97 |
16.05 |
657.8 |
5.39 |
||||
Test Item |
274 |
ISO |
2.76 |
|
|
4.36 |
3.9 |
95.5 |
CD54 |
3.65 |
0.89 |
103.5 |
3.98 |
||||
CD86 |
5.25 |
2.49 |
95.4 |
3.43 |
||||
329 |
ISO |
2.74 |
|
|
4.19 |
3.9 |
96.6 |
|
CD54 |
3.74 |
1.00 |
116.3 |
3.94 |
||||
CD86 |
5.60 |
2.86 |
109.6 |
3.50 |
||||
395 |
ISO |
2.92 |
|
|
4.28 |
3.9 |
95.7 |
|
CD54 |
3.86 |
0.94 |
109.3 |
4.05 |
||||
CD86 |
6.07 |
3.15 |
120.7 |
3.42 |
||||
474 |
ISO |
2.91 |
|
|
4.21 |
3.8 |
98.6 |
|
CD54 |
3.79 |
0.88 |
102.3 |
3.90 |
||||
CD86 |
5.30 |
2.39 |
91.6 |
3.29 |
||||
569 |
ISO |
3.01 |
|
|
4.27 |
3.8 |
98.3 |
|
CD54 |
3.96 |
0.95 |
110.5 |
3.91 |
||||
CD86 |
5.69 |
2.68 |
102.7 |
3.25 |
||||
683 |
ISO |
3.13 |
|
|
4.35 |
3.7 |
101.7 |
|
CD54 |
4.29 |
1.16 |
134.9 |
3.94 |
||||
CD86 |
7.14 |
4.01 |
153.6 |
2.76 |
||||
819 |
ISO |
3.06 |
|
|
4.28 |
3.8 |
97.7 |
|
CD54 |
4.12 |
1.06 |
123.3 |
3.98 |
||||
CD86 |
6.04 |
2.98 |
114.2 |
3.24 |
||||
983 |
ISO |
3.51 |
|
|
5.28 |
4.6 |
81.8 |
|
CD54 |
5.32 |
1.81 |
210.5 |
4.78 |
||||
CD86 |
9.46 |
5.95 |
228.0 |
3.68 |
Applicant's summary and conclusion
- Interpretation of results:
- other: skin sensitising potential based on the key event “activation of dendritic cells” of skin sensitisation AOP
- Conclusions:
- Under the conditions of the test, it can be concluded, that the test substance is predicted a sensitiser in the human Cell Line Activation Test (h-CLAT). The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation was performed.
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