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EC number: 288-914-8 | CAS number: 85940-25-6
- 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:
- 12 June 2017 - 15 August 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 2010
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Reference substance name:
- Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.
- EC Number:
- 288-914-8
- EC Name:
- Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.
- Cas Number:
- 85940-25-6
- Molecular formula:
- Molecular formula is not available
- IUPAC Name:
- Reaction product of phenol, 4-(9H-carbazol-3-ylamino)- with sodium polysulfide, leuco derivatives
- Test material form:
- solid: particulate/powder
- Details on test material:
- Test Item: Leuco Sulfur Blue 20P
Appearance: Black powder
Constituent 1
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA/Ca
- Remarks:
- CBA/Ca Ola Hsd mice
- Sex:
- female
- Details on test animals and environmental conditions:
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. Females are used because the existing database is predominantly based on females.
Number of animals: 28 animals/main test* (4 animals/treatment group, 12 shared control animals)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 12 weeks old (at start of the main test)
Body weight rangeat starting: 18.6 – 22.8 g .The weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight.
Acclimatization time: 14 days
Husbandry
Animal health: Only healthy animals (and not showing any sign of skin lesion) were used
Housing duringacclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
The same conditions were used for the dose range finding and main test animals. There were no deviations from these specifications during the experimental phase. The temperature and relative humidity were recorded daily during the acclimatization and experimental phases. Before housing the animals, the microbiological status of the room was checked.
Food and Water Supply
Animals received ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Randomization
The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups.
Study design: in vivo (LLNA)
- Vehicle:
- dimethyl sulphoxide
- Concentration:
- test item concentrations: 12.5 %, 6.25 %, 3.13 % and 1.56 % (w/w)
- No. of animals per dose:
- 28 animals/main test (4 animals/treatment group)
- Details on study design:
- Animals in the treatment groups were treated with the negative controls (vehicles), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test.
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test. All animals treated were processed and therefore no treatment group was excluded from the evaluation.
On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 µCi of
3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps.
Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % (w/v) TCA at 2-8 °C overnight
(approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4 °C and decanting the supernatants, than the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were stored at 2-8 °C. On the day of measurement samples were dispersed again using (an ultrasonic water bath), transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and loaded into the beta-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The beta-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA.
Instrument used for the measurement:
Name: Tri-Carb 3100TR, Liquid Scintillation Analyzer
Serial Number: 072971 - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- The randomization was checked by computer software [SPSS/PC+ (4.0.1)]
Results and discussion
- Positive control results:
- The positive control item (25 % (w/v) HCA in Acetone : Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation when compared to the concurrent control (SI = 10.0), thus confirming the validity of the assay.
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- EC3
- Remarks on result:
- not determinable
- Remarks:
- SI values were below 3 at all test concentrations
- Parameter:
- SI
- Value:
- 0.5
- Variability:
- p = 0.78, r = 0.22
- Test group / Remarks:
- at test item concentrations of 12.5 % (w/w)
- Parameter:
- SI
- Value:
- 1.3
- Variability:
- p = 0.78, r = 0.22
- Test group / Remarks:
- at test item concentrations of 6.25 % (w/w)
- Parameter:
- SI
- Value:
- 0.4
- Variability:
- p = 0.78, r = 0.22
- Test group / Remarks:
- at test item concentrations of 3.13 % (w/w)
- Parameter:
- SI
- Value:
- 0.3
- Variability:
- p = 0.78, r = 0.22
- Test group / Remarks:
- at test item concentrations o1.56 % (w/w)
- Cellular proliferation data / Observations:
- No significantly increased lymphoproliferation (indicated by an SI >= 3) compared to the relevant control [50 % (v/v) DMSO in water] was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.5, 1.3, 0.4 and 0.3 at test item concentrations of 12.5 %, 6.25 %, 3.13 % and 1.56 % (w/w), respectively. No significant dose-response relationship was observed (p = 0.78, r = 0.22; evaluated by linear regression using SI values).
According to the evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI >= 3) up to the maximum attainable concentration of 12.5 % (w/w) as well as the lack of a significant dose-response relationship is considered as evidence that Leuco Sulfur Blue 20P is not a skin sensitizer.
No mortality or signs of systemic toxicity were observed during the test. No significant treatment related effect on the body weights was observed in any dose group. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group.
Any other information on results incl. tables
Individual Body Weights of the Animals with Group Means, the Associated Error Termsand Body Weight Changes in the Main Test
Animal |
Dose Group |
Initial |
Terminal |
Body Weight |
Number |
Body Weight |
Body Weight |
Change |
|
(g) |
(g) |
(%) |
||
45 |
Vehicle control |
20.0 |
20.0 |
0 |
46 |
for the positive control: |
22.3 |
22.1 |
-1 |
47 |
AOO |
19.1 |
19.5 |
2 |
48 |
|
21.6 |
21.7 |
0 |
|
Mean |
20.8 |
20.8 |
0 |
|
SD |
1.5 |
1.3 |
|
49 |
Positive control: |
20.3 |
20.4 |
0 |
50 |
25 % HCA |
21.5 |
20.3 |
-6 |
51 |
in AOO |
22.8 |
22.1 |
-3 |
52 |
|
19.3 |
19.8 |
3 |
|
Mean |
21.0 |
20.7 |
-2 |
|
SD |
1.5 |
1.0 |
|
53 |
Vehicle control |
21.0 |
21.5 |
2 |
54 |
for the test item: |
21.4 |
22.0 |
3 |
55 |
50 % DMSO in water |
22.0 |
22.5 |
2 |
56 |
|
18.6 |
18.9 |
2 |
|
Mean |
20.8 |
21.2 |
2 |
|
SD |
1.5 |
1.6 |
|
89 |
Leuco Sulfur Blue 20P |
20.0 |
20.2 |
1 |
90 |
12.5 % |
22.7 |
20.7 |
-9 |
91 |
in DMSO : water |
21.7 |
20.3 |
-6 |
92 |
|
19.7 |
20.4 |
4 |
|
Mean |
21.0 |
20.4 |
-3 |
|
SD |
1.4 |
0.2 |
|
93 |
Leuco Sulfur Blue 20P |
19.8 |
20.4 |
3 |
94 |
6.25 % |
19.3 |
19.7 |
2 |
95 |
in DMSO : water |
21.7 |
21.0 |
-3 |
96 |
|
22.3 |
20.8 |
-7 |
|
Mean |
20.8 |
20.5 |
-1 |
|
SD |
1.5 |
0.6 |
|
97 |
Leuco Sulfur Blue 20P |
19.0 |
19.3 |
2 |
98 |
3.13 % |
22.1 |
20.0 |
-10 |
99 |
in DMSO : water |
19.8 |
20.0 |
1 |
100 |
|
21.9 |
20.8 |
-5 |
|
Mean |
20.7 |
20.0 |
-3 |
|
SD |
1.5 |
0.6 |
|
101 |
Leuco Sulfur Blue 20P |
19.5 |
20.0 |
3 |
102 |
1.56 % |
22.1 |
20.2 |
-9 |
103 |
in DMSO : water |
22.0 |
24.7 |
12 |
104 |
|
19.8 |
19.7 |
-1 |
|
Mean |
20.9 |
21.2 |
1 |
|
SD |
1.4 |
2.4 |
|
HCA =a-Hexylcinnamaldehyde AOO = Acetone: Olive oil 4:1 (v/v) mixture
DMSO = Dimethyl sulfoxide SD = Standard Deviation
Clinical Observations in the Main Test
Dose Group |
Animal |
Days |
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
|||||
PT |
AT |
PT |
AT |
PT |
AT |
|||||
Vehicle control for the positive control: |
45 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
46 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
47 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
48 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Positive control: 25 % HCA in AOO |
49 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
50 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
51 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
52 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Vehicle control for the test item: 50 % DMSO in water |
53 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
54 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
55 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
56 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulfur Blue 20P |
89 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
90 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
91 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
92 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulfur Blue 20P |
93 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
94 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
95 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
96 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulfur Blue 20P |
97 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
98 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
99 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
100 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulfur Blue 20P |
101 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
102 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
103 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
104 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
PT = Prior to the treatment
AT = After the treatment
HCA =a-Hexylcinnamaldehyde
AOO = Acetone: Olive oil 4:1 mixture (v/v)
DMSO = Dimethyl sulfoxide
N = Normal (no symptoms observed)
Erythema Scores in the Main Test
Dose Group |
Animal Number |
Ears |
Days |
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
||||||
PT |
AT |
PT |
AT |
PT |
AT |
||||||
Vehicle control for the positive control: |
45 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
46 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
47 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
48 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Positive control: |
49 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
50 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
51 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
52 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Vehicle control for the test item: |
53 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
54 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
55 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
56 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulfur Blue 20P |
89 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
90 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
91 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
92 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulfur Blue 20P |
93 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
94 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
95 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
96 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulfur Blue 20P |
97 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
98 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
99 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
100 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulfur Blue 20P |
101 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
102 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
103 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
104 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
L = Left R = Right PT = Prior to treatment AT = After the treatment
AOO = Acetone: Olive oil 4:1 (v/v) mixture HCA =a-Hexylcinnamaldehyde DMSO = Dimethyl sulfoxide
Visual Observations of the Lymph Nodes in the Main Test
Dose Group |
Animal |
Appearance of Lymph Nodes |
Vehicle control for the positive control: |
45 |
N |
46 |
N |
|
47 |
N |
|
48 |
N |
|
Positive control: |
49 |
Larger than the relevant control (AOO) |
50 |
Larger than the relevant control (AOO) |
|
51 |
Larger than the relevant control (AOO) |
|
52 |
Larger than the relevant control (AOO) |
|
Vehicle control for the test item: |
53 |
N |
54 |
N |
|
55 |
N |
|
56 |
N |
|
Leuco Sulfur Blue 20P |
89 |
N |
90 |
N |
|
91 |
N |
|
92 |
N |
|
Leuco Sulfur Blue 20P |
93 |
N |
94 |
N |
|
95 |
N |
|
96 |
N |
|
Leuco Sulfur Blue 20P |
97 |
N |
98 |
N |
|
99 |
N |
|
100 |
N |
|
Leuco Sulfur Blue 20P |
101 |
N |
102 |
N |
|
103 |
N |
|
104 |
N |
AOO = Acetone: Olive oil 4:1 (v/v) mixture
HCA =a-Hexylcinnamaldehyde
DMSO = Dimethyl sulfoxide
N = Normal
DPM and Stimulation Index Values for all Groups in the Main Test
Dose Group |
Measured |
Group* |
DPM/Mouse# |
Stimulation |
DPM/group |
DPM |
Index Values |
||
Vehicle control for the positive control: |
9821 |
9798.5 |
2449.6 |
1.0 |
AOO |
|
|
|
|
Positive control: |
97574 |
97551.5 |
24387.9 |
10.0 |
25 % HCA in AOO |
|
|
|
|
Vehicle control for the test item: |
11911 |
11888.5 |
2972.1 |
1.0 |
50 % DMSO in water |
|
|
|
|
Leuco Sulfur Blue 20P |
6516 |
6493.5 |
1623.4 |
0.5 |
12.5 % in DMSO : water |
|
|
|
|
Leuco Sulfur Blue 20P |
15024 |
15001.5 |
3750.4 |
1.3 |
6.25 % in DMSO : water |
|
|
|
|
Leuco Sulfur Blue 20P |
4728 |
4705.5 |
1176.4 |
0.4 |
3.13 % in DMSO : water |
|
|
|
|
Leuco Sulfur Blue 20P |
3671 |
3648.5 |
912.1 |
0.3 |
1.56 % in DMSO : water |
|
|
|
|
HCA =a-Hexylcinnamaldehyde
AOO = Acetone: Olive oil 4:1 (v/v) mixture
DMSO = Dimethyl sulfoxide
*Group DPM = measured DPMgroup- average DPMbackground
Average DPMbackground= 22.5
# Number of animals/group = 4
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item was determined not to have a skin sensitization potential.
- Executive summary:
The aim of this study according to OECD 429 was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. In general, very low solubility of the test item was observed. The test item was formulated in water resulting in a thick suspension (slurry) at a nominal concentration of 25 % (w/w) prior to dilution with an appropriate vehicle (DMSO) in a ratio of 1:1 (v/v). The 12.5 % (w/w) formulation prepared in this way was an adequately stable and homogeneous suspension for application on the dorsum of ears of animals. No significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test (12.5 % and 6.25 % (w/w) were tested). According to this the test item was examined in the main test at concentrations of 12.5 % and at 6.25 %, 3.13 % or 1.56 % (w/w; prepared by serial dilution in DMSO : water 1:1 (v/v) mixture) as suspension formulations. An appropriate positive control (a-Hexylcinnamaldehyde, HCA) and furthermore two negative control groups dosed withDMSO : water 1:1 (v/v) mixture(as vehicle control for the test groups) or AOO (as vehicle control for the positive control group) were employed.
The positive control item (25 % (w/v) HCA in Acetone : Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation when compared to the concurrent control (SI = 10.0), thus confirming the validity of the assay. No mortality or signs of systemic toxicity were observed during the test. No significant treatment related effect on the body weights was observed in any dose group. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI >= 3) compared to the relevant control [50 % (v/v) DMSO in water] was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.5, 1.3, 0.4 and 0.3 at test item concentrations of 12.5 %, 6.25 %, 3.13 % and 1.56 % (w/w), respectively. No significant dose-response relationship was observed (p = 0.78, r = 0.22; evaluated by linear regression using SI values). According to the evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI >= 3) up to the maximum attainable concentration of 12.5 % (w/w) as well as the lack of a significant dose-response relationship is considered as evidence that the test item is not a skin sensitizer.
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