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EC number: 288-896-1 | CAS number: 85940-08-5 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53015.
- 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:
- 29 August 2017 - 09 November 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:
- 22 July 2010
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 20 July 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Reference substance name:
- Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives
- EC Number:
- 288-896-1
- EC Name:
- Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives
- Cas Number:
- 85940-08-5
- Molecular formula:
- not applicable
- IUPAC Name:
- Reaction product of N-(2,4-dinitrophenyl)acetamide with 1-methyl-2,4-dinitrobenzene and polysulfide, leuco derivatives
- Test material form:
- solid: particulate/powder
- Details on test material:
- Test item: Leuco Sulphur Brown 46
Appearance: Brown powder
CAS No: 85940-08-5
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 duringthe 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 range at starting: 17.8 – 22.4 g. The weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight.
Acclimatization time: 12 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 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal
rodent activities.
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. Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive. Relevant batch number, expiry date and contents of
the diet are given in Appendix III.
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. Copies of the relevant Certificates of Analysis are maintained in
TOXI-COOP ZRT.’s archive.
Identification of Animals
The individual identification of the animals was performed by numbers on the tail. The cages were marked with identification cards, with information (at least) about study code, species, strain, sex, dose group and individual animal numbers.
Study design: in vivo (LLNA)
- Vehicle:
- dimethyl sulphoxide
- Concentration:
- 1.56, 3.13, 6.25 and 12.5 % (w/w)
- No. of animals per dose:
- 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.
Injection of 3HTdR
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.
Determination of Incorporated 3HTdR
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 % 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 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
Clinical Observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software. Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) was not calculated for the test item.
The test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive. - 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 = 15.8), 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 tested concentrations
- Parameter:
- SI
- Value:
- 2.3
- Variability:
- p = 0.01, r = 0.99
- Test group / Remarks:
- at test item concentration of 12.5 °%
- Parameter:
- SI
- Value:
- 1.6
- Variability:
- p = 0.01, r = 0.99
- Test group / Remarks:
- at test item concentration of 6.25 °%
- Parameter:
- SI
- Value:
- 1
- Variability:
- p = 0.01, r = 0.99
- Test group / Remarks:
- at test item concentrations of 3.13 °%
- Parameter:
- SI
- Value:
- 0.7
- Variability:
- p = 0.01, r = 0.99
- Test group / Remarks:
- at test item concentration of 1.56 °%
- 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 2.3, 1.6, 1.0 and 0.7 at test item concentrations of 12.5 %, 6.25 %, 3.13 % and 1.56 % (w/w), respectively. The dose-response correlation was statistically significant (p = 0.01, r = 0.99; evaluated by linear regression using SI values). According to 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) is considered as evidence that the test item 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 and the Body Weight Changes in the Dose Range Finding Test
Animal |
Dose Group |
Initial Body |
Terminal Body |
Body Weight |
Number |
Weight (g) |
Weight (g)* |
Change (%) |
|
435 |
Leuco Sulphur Brown 46 |
18.1 |
18.1 |
0 |
436 |
12.5 % in DMSO : water |
20.2 |
20.4 |
1 |
Mean |
19.2 |
19.3 |
1 |
|
437 |
Leuco Sulphur Brown 46 |
17.5 |
18.0 |
3 |
438 |
6.25 % in DMSO : water |
20.2 |
20.1 |
0 |
Mean |
18.9 |
19.1 |
1 |
*Terminal body weights were measured on Day 6.
Clinical Observations in the Dose Range Finding Test
Dose Group |
Animal Number |
Days |
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
|||||
PT |
AT |
PT |
AT |
PT |
AT |
|||||
Leuco Sulphur Brown 46 |
435 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
436 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulphur Brown 46 |
437 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
438 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
T = Treatment
PT = Prior to the treatment
AT = After the treatment
DMSO= Dimethyl sulfoxide
N = Normal (no sign of toxicity observed)
Erythema Scores in the Dose Range Finding Test
Dose Group |
Animal Number |
Ears |
Days |
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
||||||
PT |
AT |
PT |
AT |
PT |
AT |
||||||
Leuco Sulphur Brown 46 |
435 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
436 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulphur Brown 46 |
437 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
438 |
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 the treatment AT = After the treatment
DMSO= Dimethyl sulfoxide
Individual Ear Thickness Values and the Deviations from the Initial Values in the Dose Range Finding Test
Dose Group |
Animal |
Ears |
Day 1* |
Day 3$ |
Day 3 |
Day 6# |
Day 6 |
|
Number |
value (mm) |
value (mm) |
% deviation |
value (mm) |
% deviation |
|
|
435 |
L |
0.20 |
0.19 |
-5.0 |
0.19 |
-5.0 |
Leuco Sulphur Brown 46 |
R |
0.20 |
0.20 |
0.0 |
0.19 |
-5.0 |
|
12.5 % in DMSO : water |
436 |
L |
0.18 |
0.19 |
5.6 |
0.21 |
16.7 |
|
R |
0.18 |
0.19 |
5.6 |
0.20 |
11.1 |
|
|
437 |
L |
0.20 |
0.19 |
-5.0 |
0.19 |
-5.0 |
Leuco Sulphur Brown 46 |
R |
0.20 |
0.20 |
0.0 |
0.19 |
-5.0 |
|
6.25 % in DMSO : water |
438 |
L |
0.20 |
0.21 |
5.0 |
0.19 |
-5.0 |
|
R |
0.20 |
0.21 |
5.0 |
0.19 |
-5.0 |
L = Left
R = Right
DMSO= Dimethyl sulfoxide
* Ear thickness was measured prior to the first treatment.
$ Ear thickness was measured approximately 48 hours after the first treatment (prior to the third treatment).
# Ear thickness was measured at the end of the test.
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) |
(%) |
||
455 |
Vehicle control |
21.6 |
21.8 |
1 |
456 |
for the positive control: |
17.9 |
18.4 |
3 |
457 |
AOO |
20.0 |
20.5 |
3 |
458 |
|
20.8 |
20.8 |
0 |
|
Mean |
20.1 |
20.4 |
1 |
|
SD |
1.6 |
1.4 |
|
459 |
Positive control: |
20.8 |
20.8 |
0 |
460 |
25 % HCA |
17.8 |
17.6 |
-1 |
461 |
in AOO |
21.8 |
21.6 |
-1 |
462 |
|
20.0 |
20.3 |
2 |
|
Mean |
20.1 |
20.1 |
0 |
|
SD |
1.7 |
1.7 |
|
463 |
Vehicle control |
20.1 |
19.5 |
-3 |
464 |
for the test item: |
21.7 |
22.6 |
4 |
465 |
50 % DMSO in water |
20.8 |
22.7 |
9 |
466 |
|
17.9 |
18.1 |
1 |
|
Mean |
20.1 |
20.7 |
3 |
|
SD |
1.6 |
2.3 |
|
467 |
Leuco Sulphur Brown 46 |
20.7 |
20.9 |
1 |
468 |
12.5 % |
19.9 |
20.4 |
3 |
469 |
in DMSO : water |
21.9 |
21.3 |
-3 |
470 |
|
18.1 |
18.6 |
3 |
|
Mean |
20.2 |
20.3 |
1 |
|
SD |
1.6 |
1.2 |
|
471 |
Leuco Sulphur Brown 46 |
22.3 |
21.8 |
-2 |
472 |
6.25 % |
18.5 |
17.5 |
-5 |
473 |
in DMSO : water |
20.6 |
20.9 |
1 |
474 |
|
19.9 |
17.8 |
-11 |
|
Mean |
20.3 |
19.5 |
-4 |
|
SD |
1.6 |
2.2 |
|
475 |
Leuco Sulphur Brown 46 |
20.7 |
20.1 |
-3 |
476 |
3.13 % |
20.1 |
20.1 |
0 |
477 |
in DMSO : water |
18.6 |
18.7 |
1 |
478 |
|
22.2 |
21.9 |
-1 |
|
Mean |
20.4 |
20.2 |
-1 |
|
SD |
1.5 |
1.3 |
|
479 |
Leuco Sulphur Brown 46 |
20.2 |
20.4 |
1 |
480 |
1.56 % |
20.5 |
20.8 |
1 |
481 |
in DMSO : water |
22.4 |
23.0 |
3 |
482 |
|
18.8 |
18.9 |
1 |
|
Mean |
20.5 |
20.8 |
1 |
|
SD |
1.5 |
1.7 |
|
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: |
455 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
456 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
457 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
458 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Positive control: 25 % HCA in AOO |
459 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
460 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
461 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
462 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Vehicle control for the test item: 50 % DMSO in water |
463 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
464 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
465 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
466 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulphur Brown 46 |
467 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
468 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
469 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
470 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulphur Brown 46 |
471 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
472 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
473 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
474 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulphur Brown 46 |
475 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
476 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
477 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
478 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
Leuco Sulphur Brown 46 |
479 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
480 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
481 |
N |
N |
N |
N |
N |
N |
N |
N |
N |
|
482 |
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: |
455 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
456 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
457 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
458 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Positive control: |
459 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
460 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
461 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
462 |
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: |
463 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
464 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
465 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
466 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulphur Brown 46 |
467 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
468 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
469 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
470 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulphur Brown 46 |
471 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
472 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
473 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
474 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulphur Brown 46 |
475 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
476 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
477 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
478 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Leuco Sulphur Brown 46 |
479 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
480 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
481 |
L |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
R |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
482 |
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: |
455 |
N |
456 |
N |
|
457 |
N |
|
458 |
N |
|
Positive control: |
459 |
Larger than the relevant control (AOO) |
460 |
Larger than the relevant control (AOO) |
|
461 |
Larger than the relevant control (AOO) |
|
462 |
Larger than the relevant control (AOO) |
|
Vehicle control for the test item: |
463 |
N |
464 |
N |
|
465 |
N |
|
466 |
N |
|
Leuco Sulphur Brown 46 |
467 |
N |
468 |
N |
|
469 |
N |
|
470 |
N |
|
Leuco Sulphur Brown 46 |
471 |
N |
472 |
N |
|
473 |
N |
|
474 |
N |
|
Leuco Sulphur Brown 46 |
475 |
N |
476 |
N |
|
477 |
N |
|
478 |
N |
|
Leuco Sulphur Brown 46 |
479 |
N |
480 |
N |
|
481 |
N |
|
482 |
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: |
3189 |
3163.5 |
790.9 |
1.0 |
AOO |
|
|
|
|
Positive control: |
50042 |
50016.5 |
12504.1 |
15.8 |
25 % HCA in AOO |
|
|
|
|
Vehicle control for the test item: |
2602 |
2576.5 |
644.1 |
1.0 |
50 % DMSO in water |
|
|
|
|
Leuco Sulphur Brown 46 |
6067 |
6041.5 |
1510.4 |
2.3 |
12.5 % in DMSO : water |
|
|
|
|
Leuco Sulphur Brown 46 |
4209 |
4183.5 |
1045.9 |
1.6 |
6.25 % in DMSO : water |
|
|
|
|
Leuco Sulphur Brown 46 |
2663 |
2637.5 |
659.4 |
1.0 |
3.13 % in DMSO : water |
|
|
|
|
Leuco Sulphur Brown 46 |
1707 |
1681.5 |
420.4 |
0.7 |
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 DPMbackgroundAverage DPMbackground= 25.5
# Number of animals/group = 4
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item was shown to have no skin sensitization potential in the Local Lymph Node Assay.
- Executive summary:
The aim of this study was to evaluate the skin sensitization potential of the test following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. Preliminary tests were performed to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item was evaluated in N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO) at 25 % and 12.5 % (w/w) concentrations (based on previous studies performed for test items with similar chemical characteristics). Limited solubility of the test item has been observed in both vehicles and the formulations were not adequately homogeneous for a proper application. According to the Sponsor’s request 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 with DMSO : 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 = 15.8), 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 a 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 2.3, 1.6, 1.0 and 0.7 at test item concentrations of 12.5 %, 6.25 %, 3.13 % and 1.56 % (w/w), respectively. The dose-response correlation was statistically significant (p = 0.01, r = 0.99; evaluated by linear regression using SI values). According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by a SI >= 3) up to the maximum attainable concentration of 12.5 % (w/w) is considered as evidence that the test item is not a skin sensitizer. In conclusion, under the conditions of the present assay, the test item tested at the maximum feasible concentration of 12.5 % (w/w) and also at concentrations of 6.25 %, 3.13 % or 1.56 % (w/w) as adequate homogeneous formulations was shown to have no skin sensitization potential in the Local Lymph Node Assay.
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