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EC number: 835-272-7 | CAS number: 256374-76-2
- 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:
- 19 March 2014 to 18 June 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- OECD Guidelines for Testing of Chemicals No. 429 “Skin Sensitisation: Local Lymph Node Assay” (22 July 2010)
- Deviations:
- yes
- Remarks:
- See "any other information" for details
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L 142, 31/05/2008) as amended by Commission Regulation (EU) No 640/2012 of 6 July 2012 (Official Journal L 193, 20/07/2012)
- Deviations:
- yes
- Remarks:
- see "any other information" for details
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
Test material
- Reference substance name:
- 2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one
- EC Number:
- 835-272-7
- Cas Number:
- 256374-76-2
- Molecular formula:
- C12H26N2O6P2
- IUPAC Name:
- 2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one
- Test material form:
- solid: particulate/powder
- Details on test material:
- Name
Name: Reaction products of ethane-1,2-diamine, phosphoryl=trichloride and 2,2-dimethylpropane-1,3-diol which makes N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane=2-oxide-2-yl)ethane-1,2-diamine as a main component
Other name: SH-0850
CAS number: 256374-76-2 (main component)
Structural formula
Molecular formula: C12H26N2O6P2 (main component)
Molecular weight: 356.29 (main component)
Provided sample
Purity of the test substance: 100%
Lot number: SK-241002
Physical-chemical properties
Solubility in water: Less than 0.03% (w/w) by visual observation
Melting point: 277 °C
Appearance at normal temperatures: White powder
Storage condition
The test substance was stored in a dark place at room temperature.
Precaution for handling
Protective gloves, mask, glasses and clothes were put on in order to avoid contacts with skin or eyes and inhalation.
Constituent 1
- Specific details on test material used for the study:
- No further details specified in the study report.
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- CBA:J
- Sex:
- female
- Details on test animals and environmental conditions:
- Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER
Route des Chènes Secs B.P. 4105
53940 LE GENEST-ST-ISLE, France
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group (Experiments I-II.) or 5 animals / group (Experiment III.)
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 10 weeks old (Experiment I.); 11 weeks old (Experiment II.); 9 weeks old (Experiment III.). (Animals were age-matched, within one week in each test)
Body weight range at starting: 20.8-21.7 grams (Experiment I.); 20.1-22.5 grams (Experiment II.); 21.6-24.5 grams (Experiments III.) (The weight variation in animals of the study did not exceed ± 20 % of the mean weight in each test.)
Acclimatization time: at least 5 days
Note: In the preliminary experiment, mice of 8 weeks of age (18.7-19.9 grams) were used.
Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 17.8 - 25.3°C
Relative humidity: 30 - 75 %
Ventilation: 15-20 air exchange/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (non-radioactive phase): 244/3 (preliminary experiment); 244/4 (Experiment I.); 242/1 (Experiment II.); 244/3 (Experiment III.)
Room/Cabinet (radioactive phase): 139 – 140
Food and feeding
Animals received ssniff SM R/M-15mm "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 186 0298, Expiry date: 31 May 2014; and Batch number: 672 1467, Expiry date: 30 November 2014) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water supply
Animals received tap water from the municipal supply from 500 mL bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at CiToxLAB Hungary Ltd.
Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-FASERN Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study. Nest building material (Cotton Rolls produced by AsBe-wood GmbH, Berta-von-Suttner-Allee 4, 21614 Buxtehude, Germany or by DC Dental Central Grosshandelges. mbH, Carl-Zeiss Str. 2, D-22946 Trittau, Germany) was also provided to the animals.
Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number.
The animals were randomised and allocated to the experimental groups in each experiment. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.
Study design: in vivo (LLNA)
- Vehicle:
- methyl ethyl ketone
- Concentration:
- 50%, 25% and 10%
- No. of animals per dose:
- 2 animals/dose
- Details on study design:
- Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/J Rj mice using two doses (2 animals/dose) at test item concentrations of 50 and 25% (w/v) in MEK. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
In the Preliminary Irritation / Toxicity Test, no mortality or clinical signs of systemic toxicity were observed. Test item precipitate was observed on the ears of the animals in the 50 and 25% (w/v) dose groups on Days 1-3. No marked body weight loss (>5%) was observed in the experimental animals.
Ear thickness of the animals was measured by using a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. Both ears of each mouse were observed for erythema and scored.
The draining auricular lymph nodes of the animals were visually examined: the appearance of the lymph nodes was normal in the test item treated groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 50% (w/v) dose was considered to be acceptable as the highest examined concentration for the main test.
Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. 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.
OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the each test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the lymph nodes were collected in Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. In Experiments I-II., the nodes of mice from each test group were pooled; in Experiment III., the lymph nodes of each animal were processed individually.
Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) 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 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes (Experiments I-II.) or for each animal (Experiment III.).
Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- DPM was measured for each sample (for each pooled group of nodes in Experiments I-II., or for each animal in Experiment III.). The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) for each sample following the industry standard for data presentation.
Stimulation index (SI = mean DPN value of a treated group divided by the mean DPN value of the negative control group) for each treatment group was also calculated in all experiments. A stimulation index of 3 or greater is an indication of a positive result.
In Experiment III., the use of the individual approach to calculate SI made the use of a statistical analysis available*. The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test.
Results and discussion
- Positive control results:
- No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index values of 26.5, 4.4 and 15.2 in Experiments I, II and III, respectively) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- SI
- Value:
- 0.7
- Test group / Remarks:
- Test Item 50% w/v
- Key result
- Parameter:
- SI
- Value:
- 0.9
- Test group / Remarks:
- Test item 25% w/v
- Key result
- Parameter:
- SI
- Value:
- 1.1
- Test group / Remarks:
- Test Item 10% w/v
- Cellular proliferation data / Observations:
- Based on the results of the main test (Experiment I.), an additional experiment (Experiment II.) was performed for clarification. The additional experiment was performed using the same treatment groups and same experimental conditions.
Based on the results of the second main test, another additional experiment (Experiment III.) was performed for clarification. The additional experiment was performed using the same treatment groups (but 5 animals/group) and same experimental conditions except that individual approach was used in the radioactive proliferation measurement.
In each experiment, the appearance of the lymph nodes was normal in the negative (vehicle) control group and for most of the animals in the test item treated groups. Larger than normal lymph nodes was observed in the positive control groups, and slightly enlarged lymph nodes were recorded for one test item treated animal in the 50% (w/v) dose group in Experiment I. (subjective judgement by analogy with observations of former experiments).
In Experiment I., the stimulation index values were 3.1, 1.0 and 3.3 at concentrations of 50, 25 and 10% (w/v), respectively.
In Experiment II., the stimulation index values were 1.1, 0.7 and 1.1. at concentrations of 50, 25 and 10% (w/v), respectively.
In Experiment III., the stimulation index values were 0.7, 0.9 and 1.1 at concentrations of 50, 25 and 10% (w/v), respectively.
CLINICAL OBSERVATIONS
In Experiment I., no mortality was observed and no signs of systemic toxicity were detected. Test item precipitate / minimal amount of test item precipitate was detected on the ears of the animals in the 50, 25 and 10% (w/v) dose groups on Days 1-3.
In Experiment II., no mortality was observed and no signs of systemic toxicity were detected. Test item precipitate / minimal amount of test item precipitate was detected on the ears of one or more animals in the 50, 25 and 10% (w/v) dose groups on Days 1-3.
In Experiment III., no mortality was observed and no signs of systemic toxicity were detected. Test item precipitate / minimal amount of test item precipitate was detected on the ears of one or more animals in the 50, 25 and 10% (w/v) dose groups on Days 1-3.
BODY WEIGHT MEASUREMENTS
In Experiment I., no marked body weight loss (>5%) was recorded for the experimental animals except of one negative control animal and one animal in the 50% (w/v) dose group, but these values were considered as individual variability and they did not affect the results of the study.
In Experiment II., no marked body weight loss (>5%) was recorded for the experimental animals except of one animal in the 50 and 10% (w/v) dose groups and one animal in the positive control group. However, these values were considered as individual variability and they did not affect the results of the study.
In Experiment III., no marked body weight loss (>5%) was recorded for the experimental animals except of one animal in the 50 and 10% (w/v) dose groups. However, these values were considered as individual variability and they did not affect the results of the study.
Any other information on results incl. tables
Individual Body Weights for all Animals with group Means (Experiment I)
Animal Number |
Identity Number |
Test Group Name |
Initial Body Weight (g) |
Terminal Body Weight* (g) |
Change # (%) |
5641 5651 5687 5691 |
1 2 3 4 |
Negative (vehicle) control (MEK)
Mean |
21.2 21.6 21.3 20.9 21.3 |
19.9 22.1 21.0 21.1 21.0 |
-6.1 2.3 -1.4 1.0 -1.1 |
5640 5649 5654 5706 |
5 6 7 8 |
SH-0850 50% (w/v) in MEK
Mean |
21.1 21.7 21.1 20.9 21.2 |
21.3 22.5 21.9 19.2 21.2 |
0.9 3.7 3.8 -8.1 0.1 |
5715 5656 5659 5646 |
9 10 11 12 |
SH-0850 25% (w/v) in MEK
Mean |
21.6 21.1 21.6 20.8 21.3 |
20.9 21.8 21.5 20.3 21.1 |
-3.2 3.3 -0.5 -2.4 -0.7 |
5662 5643 5653 5688 |
13 14 15 16 |
SH-0850 10% (w/v) in MEK
Mean |
21.2 21.5 21.6 20.8 21.3 |
20.4 21.9 20.7 20.9 21.0 |
-3.8 1.9 -4.2 0.5 -1.4 |
5676 5672 5669 5663 |
17 18 19 20 |
Positive control 25% (w/v) HCA in MEK
Mean |
21.6 21.2 21.4 20.9 21.3 |
23.9 23.2 22.8 21.4 22.8 |
10.6 9.4 6.5 2.4 7.3 |
*: Terminal body weights were measured on Day 6
#: (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100
Individual Body Weights for all Animals with group Means (Experiment II)
Animal Number |
Identity Number |
Test Group Name |
Initial Body Weight (g) |
Terminal Body Weight* (g) |
Change # (%) |
6278 6333 6345 6328 |
1 2 3 4 |
Negative (vehicle) control (MEK)
Mean |
22.4 21.2 20.4 20.6 21.2 |
21.4 22.1 22.0 20.8 21.6 |
-4.5 4.2 7.8 1.0 2.1 |
6318 6343 6321 6334 |
5 6 7 8 |
SH-0850 50% (w/v) in MEK
Mean |
22.5 20.9 20.1 20.3 21.0 |
22.4 21.4 18.9 20.7 20.9 |
-0.4 2.4 -6.0 2.0 -0.5 |
6320 6329 6326 6336 |
9 10 11 12 |
SH-0850 25% (w/v) in MEK
Mean |
22.5 20.8 20.1 20.7 21.0 |
23.0 21.4 21.8 21.4 21.9 |
2.2 2.9 8.5 3.4 4.2 |
6341 6323 6331 6339 |
13 14 15 16 |
SH-0850 10% (w/v) in MEK
Mean |
21.5 21.4 20.9 21.6 21.4 |
20.6 22.2 20.8 19.4 20.8 |
-4.2 3.7 -0.5 -10.2 -2.8 |
6324 6340 6346 6325 |
17 18 19 20 |
Positive control 25% (w/v) HCA in MEK
Mean |
21.5 21.6 21.4 21.0 21.4 |
19.4 22.8 22.4 21.5 21.5 |
-9.8 5.6 4.7 2.4 0.7 |
*: Terminal body weights were measured on Day 6
#: (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100
Individual Body Weights for all Animals with group Means (Experiment I)
Animal Number |
Identity Number |
Test Group Name |
Initial Body Weight (g) |
Terminal Body Weight* (g) |
Change # (%) |
6447 6459 6487 6496 6451 |
1 2 3 4 5 |
Negative (vehicle) control (MEK)
Mean |
24.2 23.7 22.8 22.5 21.9 23.0 |
23.3 22.8 23.3 22.1 22.7 22.8 |
-3.7 -3.8 2.2 -1.8 3.7 -0.7 |
6497 6456 6453 6491 6475 |
6 7 8 9 10 |
SH-0850 50% (w/v) in MEK
Mean |
23.9 23.7 23.2 22.1 22.4 23.1 |
22.4 23.5 22.3 22.3 22.5 22.6 |
-6.3 -0.8 -3.9 0.9 0.4 -1.9 |
6501 6454 6458 6486 6472 |
11 12 13 14 15 |
SH-0850 25% (w/v) in MEK
Mean |
23.8 21.6 23.7 23.4 23.0 23.1 |
24.1 21.5 23.8 22.9 22.3 22.9 |
1.3 -0.5 0.4 -2.1 -3.0 -0.8 |
6461 6476 6482 6478 6494 |
16 17 18 19 20 |
SH-0850 10% (w/v) in MEK
Mean |
24.3 23.7 23.7 22.0 22.4 23.2 |
22.5 23.4 23.6 22.3 22.6 22.9 |
-7.4 -1.3 -0.4 1.4 0.9 -1.4 |
6468 6485 6481 6483 6467 |
21 22 23 24 25 |
Positive control 25% (w/v) HCA in MEK
Mean |
24.5 23.1 22.7 22.2 22.5 23.0 |
24.9 23.6 23.5 21.9 23.5 23.5 |
1.6 2.2 3.5 -1.4 4.4 2.1 |
*: Terminal body weights were measured on Day 6
#: (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100
DPM, DPN and Stimulation Index Values for all Groups (Experiment I)
Test Group Name |
Measured DPM/group |
DPM |
Number of lymph nodes |
DPN |
Stimulation Index |
Background (5% (w/v) TCA) |
34 32 |
- |
- |
- |
- |
Negative (vehicle) control (MEK) |
1198 |
1165.0 |
8 |
145.6 |
1.0 |
SH-0850 50% (w/v) in MEK |
3683 |
3650.0 |
8 |
456.3 |
3.1 |
SH-0850 25% (w/v) in MEK |
1200 |
1167.0 |
8 |
145.9 |
1.0 |
SH-0850 10% (w/v) in MEK |
3925 |
3892.0 |
8 |
486.5 |
3.3 |
Positive control (25% (w/v) HCA in MEK) |
30858 |
30825.0 |
8 |
3853.1 |
26.5 |
DPM, DPN and Stimulation Index Values for all Groups (Experiment II)
Test Group Name |
Measured DPM/group |
DPM |
Number of lymph nodes |
DPN |
Stimulation Index |
Background (5% (w/v) TCA) |
38 37 |
- |
- |
- |
- |
Negative (vehicle) control (MEK) |
2989 |
2951.5 |
8 |
368.9 |
1.0 |
SH-0850 50% (w/v) in MEK |
3174 |
3136.5 |
8 |
392.1 |
1.1 |
SH-0850 25% (w/v) in MEK |
2046 |
2008.5 |
8 |
251.1 |
0.7 |
SH-0850 10% (w/v) in MEK |
3255 |
3217.5 |
8 |
402.2 |
1.1 |
Positive control (25% (w/v) HCA in MEK) |
13018 |
12980.5 |
8 |
1622.6 |
4.4 |
DPM, DPN and Stimulation Index Values for all Groups (Experiment III)
Test Group Name |
ID Number |
Measured DPM |
DPM |
Number of Lymph Nodes |
DPN |
Group DPN |
Stimulation Index |
Background (5% (w/v) TCA) |
- |
30 35 |
- |
- |
- |
- |
- |
Negative control (MEK) |
|
187 335 259 141 140 |
154.5 302.5 226.5 108.5 107.5 |
2 2 2 2 2 |
77.3 151.3 113.3 54.3 53.8 |
90.0 |
1.0 |
SH-0850 50% (w/v) in MEK |
|
102 194 198 240 93 |
69.5 161.5 165.5 207.5 60.5 |
2 2 2 2 2 |
34.8 80.8 82.8 103.8 30.3 |
66.5 |
0.7 |
SH-0850 25% (w/v) in MEK |
|
171 248 171 202 148 |
138.5 215.5 138.5 169.5 115.5 |
2 2 2 2 2 |
69.3 107.8 69.3 84.8 57.8 |
77.8 |
0.9 |
SH-0850 10% (w/v) in MEK |
|
272 203 159 194 286 |
239.5 170.5 126.5 161.5 253.5 |
2 2 2 2 2 |
119.8 85.3 63.3 80.8 126.8 |
95.2 |
1.1 |
Positive control (25% HCA in MEK) |
|
1071 2776 1978 3706 4333 |
1038.5 2743.5 1945.5 3673.5 4300.5 |
2 2 2 2 2 |
519.3 1371.8 972.8 1836.8 2150.3 |
1370.2 |
15.2** |
Note:
** = Significant (p<0.01, Mann-Whitney U-test versus negative control)
Results of the Preliminary Irritation / Toxicity Test
Individual Body Weights for all Animals with Group Means
Animal Number |
Identity Number |
Test Group Name |
Initial Body Weight (g) |
Terminal Body Weight* (g) |
Change# (%) |
5333 5345 |
1 2 |
50% (w/v) 50% (w/v) Mean |
19.9 19.0 19.5 |
20.8 19.8 20.3 |
4.5 4.2 4.4 |
5339 5350 |
3 4 |
25% (w/v) 25% (w/v) Mean |
19.2 18.7 19.0 |
20.2 17.8 19.0 |
5.2 -4.8 0.3 |
Notes:
*: Terminal body weights were measured on Day 6.
#: (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100
Individual Ear Thickness for all Animals
Animal Number |
Identity Number |
Test Group Name |
Ear Thickness on Day 1 (mm) |
Ear Thickness on Day 3 (mm) |
Ear Thickness on Day 6 (mm) |
Biopsy weight on Day 6 (mg) |
5333 5345 5339 5350 |
1 2 3 4 |
50% (w/v) 50% (w/v) 25% (w/v) 25% (w/v) |
0.20 / 0.21 0.20 / 0.20 0.20 / 0.20 0.20 / 0.20 |
0.21 / 0.21 0.22 / 0.21 0.21 / 0.20 0.20 / 0.20 |
0.21 / 0.22 0.22 / 0.21 0.21 / 0.20 0.20 / 0.20 |
13.13 13.22 14.18 12.92 |
Notes:
Ear thickness measurements: (data of right ear) / (data of left ear)
General historical control range for biopsy: 11.92-22.53 mg. Positive response is over 28.16 mg.
Summarized Clinical Observations
Period |
Group |
Animal No. |
Identity No. |
Clinical observations |
DAY 1 |
50% (w/v) |
1 |
5333 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0 |
2 |
5345 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0 |
||
25% (w/v) |
3 |
5339 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0 |
|
4 |
5350 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0 |
||
DAY 2 |
50% (w/v) |
1 |
5333 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0 |
2 |
5345 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0 |
||
25% (w/v) |
3 |
5339 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0 |
|
4 |
5350 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0 |
||
DAY 3 |
50% (w/v) |
1 |
5333 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0 |
2 |
5345 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0 |
||
25% (w/v) |
3 |
5339 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0 |
|
4 |
5350 |
Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0 |
||
DAY 4 |
50% (w/v) |
1 |
5333 |
Symptom-free, ES: 0 |
2 |
5345 |
Symptom-free, ES: 0 |
||
25% (w/v) |
3 |
5339 |
Symptom-free, ES: 0 |
|
4 |
5350 |
Symptom-free, ES: 0 |
||
DAY 5 |
50% (w/v) |
1 |
5333 |
Symptom-free, ES: 0 |
2 |
5345 |
Symptom-free, ES: 0 |
||
25% (w/v) |
3 |
5339 |
Symptom-free, ES: 0 |
|
4 |
5350 |
Symptom-free, ES: 0 |
||
DAY 6 |
50% (w/v) |
1 |
5333 |
Symptom-free, ES: 0 |
2 |
5345 |
Symptom-free, ES: 0 |
||
25% (w/v) |
3 |
5339 |
Symptom-free, ES: 0 |
|
4 |
5350 |
Symptom-free, ES: 0 |
Notes:
The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.
ES: Erythema score
*: test item precipitate, **: minimal amount of test item precipitate
Summarized Clinical Observations
Summarized Clinical Observations (Experiment I)
Group |
Identity No. |
CLINICAL OBSERVATIONS |
|||||
DAY 1 |
DAY 2 |
DAY 3 |
DAY 4 |
DAY 5 |
DAY 6 |
||
Negative control (MEK) |
5641
5651
5687
5691
|
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 50% (w/v) in MEK |
5640
5649
5654
5706
|
BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* |
BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* |
BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 25% (w/v) in MEK |
5715
5656
5659
5646
|
BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* |
BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* |
BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 10% (w/v) in MEK |
5662
5643
5653
5688
|
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Positive control (25% (w/v) HCA in MEK) |
5676
5672
5669
5663 |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Notes:
BT: before treatment, AT: after treatment
*: test item precipitate, **: minimal amount of test item precipitate
Summarized Clinical Observations (Experiment II)
Group |
Identity No. |
CLINICAL OBSERVATIONS |
|||||
DAY 1 |
DAY 2 |
DAY 3 |
DAY 4 |
DAY 5 |
DAY 6 |
||
Negative control (MEK) |
6278
6333
6345
6238
|
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 50% (w/v) in MEK |
6318
6343
6321
6334
|
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 25% (w/v) in MEK |
6320
6329
6326
6336
|
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 10% (w/v) in MEK |
6341
6323
6331
6339
|
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free** |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Positive control (25% (w/v) HCA in MEK) |
6324
6340
6346
6325 |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Notes:
BT: before treatment, AT: after treatment
*: test item precipitate, **: minimal amount of test item precipitate
Summarized Clinical Observations (Experiment I)
Group |
Identity No. |
CLINICAL OBSERVATIONS |
|||||
DAY 1 |
DAY 2 |
DAY 3 |
DAY 4 |
DAY 5 |
DAY 6 |
||
Negative control (MEK) |
6447
6459
6487
6496
6451
|
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 50% (w/v) in MEK |
6497
6456
6453
6491
6475
|
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 25% (w/v) in MEK |
6501
6454
6458
6486
6472
|
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free* BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free* |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free** |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
SH-0850 10% (w/v) in MEK |
6461
6476
6482
6478
6494
|
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free** BT: symptom-free AT: symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Positive control (25% (w/v) HCA in MEK) |
6468
6485
6481
6483
6467 |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Symptom-free
Symptom-free
Symptom-free
Symptom-free
Symptom-free |
Notes:
BT: before treatment, AT: after treatment
*: test item precipitate, **: minimal amount of test item precipitate
Historical Control Data
(Updated in 04 July 2013)
Historical Control Data of the Positive and Negative Controls
|
Vehicles |
||||||||
Acetone-Olive oil (AOO) |
1% Pluronic PE9200 in water |
Methyl ethyl ketone (MEK) |
|||||||
DPN values |
SI value |
DPN values |
SI value |
DPN values |
SI value |
||||
Control |
HCA 25% |
HCA 25% |
Control |
HCA 25% |
HCA 25% |
Control |
HCA 25% |
HCA 25% |
|
Average |
172.6 |
1519.0 |
10.7 |
118.63 |
839.8 |
8.1 |
118.8 |
727.6 |
8.5 |
Range: Min: Max: |
36.4 586.9 |
304.9 3300.5 |
3.2 29.2 |
21.4 469.6 |
190.6 3069.4 |
3.1 55.1 |
72.2 232.5 |
412.6 1042.6 |
4.9 12.0 |
Number of cases |
100 |
72 |
72 |
82 |
55 |
55 |
4 |
2 |
2 |
|
Vehicles |
||||||||
N,N-Dimethylformamide (DMF) |
Dimethyl sulfoxide (DMSO) |
n-Hexane: Olive Oil ((HOO) |
|||||||
DPN values |
SI value |
DPN values |
SI value |
DPN values |
SI value |
||||
Control |
HCA 25% |
HCA 25% |
Control |
HCA 25% |
HCA 25% |
Control |
HCA 25% |
HCA 25% |
|
Average |
151.2 |
1917.6 |
13.5 |
249.4 |
2166.8 |
9.5 |
125.9 |
914.3 |
6.7 |
Range: Min: Max: |
20.8 423.1 |
350.9 4438.9 |
3.8 75.7 |
101.6 553.3 |
1052.8 5291.3 |
4.2 24.1 |
81.1 165.9 |
490.0 1296.4 |
5.0 8.3 |
Number of cases |
91 |
43 |
43 |
27 |
19 |
19 |
10 |
5 |
5 |
|
Vehicles |
|||||||
Propylene glycol (PG) |
Absolute ethanol: Distilled water 70:30 mixture (EtOH) |
|||||||
DPN values |
SI value |
DPN Values |
SI values |
|||||
Control |
HCA 25% |
HCA 25% |
Control |
HCA 10% |
HCA 25% |
HCA 10% |
HCA 25% |
|
Average |
131.8 |
1317.3 |
12.2 |
123.1 |
1264.2 |
3805.0 |
17.3 |
36.8 |
Range: Min: Max: |
50.6 288.8 |
510.4 2280.2 |
3.7 27.9 |
52.6 357.6 |
1214.8 1313.5 |
2178.8 9207.1 |
17.1 17.4 |
25.7 54.3 |
Number of cases |
22 |
16 |
16 |
6 |
2 |
5 |
2 |
5 |
HCA = α-Hexylcinnamaldehyde
SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.
DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.
In case of individual approach, SI values were calculated from the mean DPN values of the group.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The study required some repeat experiments; in Experiment I. the dose results did not follow a conventional dose response, in Experiment II. the negative control value was outside the expected historical range. In Experiment III., the stimulation index values were 0.7, 0.9 and 1.1 at concentrations of 50, 25 and 10% (w/v), respectively.
In conclusion, under the conditions of the present assay SH-0850, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay. - Executive summary:
The aim of this study was to determine the skin sensitisation potential of SH-0850 following dermal exposure. The study was performed with vertebrate animals as no regulatory in vitro alternative is available. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.
Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in Methyl ethyl ketone (MEK). The highest achievable concentration was 50% (w/v).
The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using two doses (50 and 25% (w/v) concentrations) in the selected vehicle. Based on the observations recorded in the preliminary test, the 50% (w/v) concentration was selected as top dose for the main tests.
In the main tests, female CBA/J Rj mice were allocated to five groups (in Experiments I-II. four animals were used in each group; in Experiment III. five animals were used for each group):
- three groups received SH-0850 (formulated in MEK) at 50, 25 and 10% (w/v) concentrations,
- the negative control group received the vehicle (MEK),
- the positive control group received 25% (w/v) HCA (dissolved in MEK).
The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).
No mortality or signs of systemic toxicity were observed in the performed experiments. Test item precipitate / minimal amount of test item precipitate was detected on the ears of one or more animals in the 50, 25 and 10% (w/v) dose groups on Days 1-3 in each experiment. There was no treatment related effect on the body weight in any of the experiments. There was no indication of irritation at the site of application.
The study required some repeat experiments; in Experiment I. the dose results did not follow a conventional dose response, in Experiment II. the negative control value was outside the expected historical range. In Experiment III., the stimulation index values were 0.7, 0.9 and 1.1 at concentrations of 50, 25 and 10% (w/v), respectively.
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 26.5, 4.4 and 15.2 in Experiments I, II, and III, respectively) was noted for the positive control chemical, this result confirmed the validity of the assay.
In conclusion, under the conditions of the present assay, SH-0850, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
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