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EC number: 940-673-6 | CAS number: 1391764-61-6
- 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 vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- December 2013 to March 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: According to OECD guideline under GLP
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- Principles of method if other than guideline:
- Not relevant
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Reference substance name:
- K-36
- IUPAC Name:
- K-36
- Reference substance name:
- N,N-dibutyl-4-[4-(4-chlorophenyl)-3,6-dioxo-2H,3H,5H,6H-pyrrolo[3,4-c]pyrrol-1-yl]benzamide
- Cas Number:
- 1391764-61-6
- Molecular formula:
- C27H28ClN3O3
- IUPAC Name:
- N,N-dibutyl-4-[4-(4-chlorophenyl)-3,6-dioxo-2H,3H,5H,6H-pyrrolo[3,4-c]pyrrol-1-yl]benzamide
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
Constituent 1
Constituent 2
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- other: CBA/CaOlaHsd
- Sex:
- female
- Details on test animals and environmental conditions:
- Number of animals for
the pre-test: 2 females
Number of animals for
the main study: 24 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 2 (1 group for the test item, 1 group for positive control)
Number of positive control groups: 1
Age: Pre-test: 10 - 11 weeks (beginning of treatment)
Main study: 8 - 9 weeks (beginning of treatment)
Body weight: see Appendix 1 and 2
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Study design: in vivo (LLNA)
- Vehicle:
- propylene glycol
- Concentration:
- The highest test item concentration, which could be technically used was a 10% suspension in PG.
- No. of animals per dose:
- 4 females (nulliparous and non-pregnant)
- Details on study design:
- Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5 and 10% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface ( 8 mm) of each ear once daily for three consecutive days. Two further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Determination of Ear Thickness
Prior to the first and third application of the test item (study days 1 and 3) and prior to treatment with 3HTdR (study day 6), the ear thickness was determined using a micrometer.
Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.6 µCi of 3H-methyl thymidine (equivalent to 78.4 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
Determination of Ear Weights
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
Interpretation of raw data
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
The ANOVA (Dunnett-test) was conducted on the ear weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group.
Also, a statistical analysis was conducted on the ear thickness values to assess whether a statistically significant increase in ear thickness could be observed when comparing the values measured on day 1 prior to application with the values measured on days 3 or 6 in the respective test item groups or within the vehicle control group.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used.
However, both biological and statistical significance were considered together.
Results and discussion
- Positive control results:
- The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2013.
In vivo (LLNA)
Resultsopen allclose all
- Parameter:
- SI
- Remarks on result:
- other: In this study Stimulation Indices (S.I.) of 1.66, 1.39 and 1.72 were determined with the test item at concentrations of 2.5, 5 and 10% formulated in PG, respectively.
- Parameter:
- other: disintegrations per minute (DPM)
- Remarks on result:
- other: see Remark
- Remarks:
- Concentration % Group Measurement DPM Calculation Result DPM-BGa) number of lymph nodes DPM per lymph nodeb) S.I. --- BG I 19 --- --- --- --- --- BG II 45 --- --- --- --- 0 1 2604 2572.0 8 321.5 1.00 2.5 2 4292 4260.0 8 532.5 1.66 5 3 3603 3571.0 8 446.4 1.39 10 4 4450 4418.0 8 552.3 1.72 0 5 6322 6290.0 8 786.3 1.00 25 6 34535 34503.0 8 4312.9 5.49
Any other information on results incl. tables
Calculation and Results of Individual Data
Vehicle: PG
Concentration % |
Group |
Measurement DPM |
Calculation |
Result |
||
DPM-BGa) |
number of lymph nodes |
DPM per lymph nodeb) |
S.I. |
|||
--- |
BG I |
19 |
--- |
--- |
--- |
--- |
--- |
BG II |
45 |
--- |
--- |
--- |
--- |
0 |
1 |
2604 |
2572.0 |
8 |
321.5 |
1.00 |
2.5 |
2 |
4292 |
4260.0 |
8 |
532.5 |
1.66 |
5 |
3 |
3603 |
3571.0 |
8 |
446.4 |
1.39 |
10 |
4 |
4450 |
4418.0 |
8 |
552.3 |
1.72 |
0 |
5 |
6322 |
6290.0 |
8 |
786.3 |
1.00 |
25 |
6 |
34535 |
34503.0 |
8 |
4312.9 |
5.49 |
1 = Control Group (PG)
2-4= Test Groups (K-36)
5 = Control Group for Positive Control (acetone/olive oil (4+1, v/v))
6 = Positive Control Group (Alpha-Hexylcinnamaldehyde; HCA)
a) = The mean value was taken from the figures BG I and BG II
b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Remarks:
- Migrated information
- Conclusions:
- The test item K-36 was not a skin sensitiser under the test conditions of this study.
- Executive summary:
In the study the test item K-36 formulated in PG was assessed for its possible skin sensitising
potential.
For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5
and 10%. The highest concentration tested was the highest concentration that could be technically
achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the
pre-experiment.
The animals did not show any signs of systemic toxicity during the course of the study and no
cases of mortality were observed. The observation of skin irritation was not possible due to
inherent color of the test item.
On day 3 and 4, the animals treated with positive control substance showed an erythema of the
ear skin (Score 1).
In the groups treated with a test item concentration of 2.5 and 10%, a statistically significant
increase (p < 0.05) in ear weight was observed in comparison to the values of the vehicle control
group. However, this increase was considered to be not biologically relevant.
In this study Stimulation Indices (S.I.) of 1.66, 1.39 and 1.72 were determined with the test item
at concentrations of 2.5, 5 and 10% formulated in PG, respectively.
The test item K-36 was not a skin sensitiser under the test conditions of this study.
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