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EC number: - | CAS number: -
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 17 March, 2020 to 29 april, 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
- Version / remarks:
- adopted 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA): BrdU-ELISA
Test material
Reference
- Name:
- Unnamed
- Type:
- Constituent
- Test material form:
- solid
In vivo test system
Test animals
- Species:
- mouse
- Strain:
- Balb/c
- Sex:
- female
- Details on test animals and environmental conditions:
- Test System: BALB/cMice
Scientific Name : Mus musculus
Justification: As this test system show good sensitization response to the positive control.
Source: Animal Breeding, RCC Laboratories India Private Limited, Genome Valley, Turkapally, Shameerpet (Manda!), Ranga Reddy District, Hyderabad - 500 078 India.
Pretest
Total number of Animals used: 8 Females (females were nulliparous and non-pregnant)
Main Study
Number of Animals per Group: 5 Females (females were nulliparous and non-pregnant)
Number of Groups: 6
Total Number of Animals: 30
Age when Treated: 11- 13 Weeks
Body weight when treated : Females: 21.72 to 24.52 g
Identification: By unique cage number and individual animal numbers marked with indelible marker pen on the tail. The animals were marked (towards the tip of tail) with red color for the temporary animal numbers at start of acclimatization. The animals were marked with black color for permanent animal numbers (towards the base of tail) before the start of test item administration.
Randomization: Animals were selected and grouped based on stratified randomization by using body weights. Computerized statistical analysis was used for randomization
Acclimatization: Under laboratory conditions for 07 days, after veterinary examination. Only animals without visible signs of illness were used for the study.
Environment
The animal room was air-conditioned with adequate (above 10) air changes per hour. The air was continuously monitored for temperature and relative humidity. The ranges for room temperature and relative humidity were 22.9 to 24.5°C and 50 to 65%, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark.
Accomodation
Individually in Polycarbonate cages (approximate internal dimensions of 214 mm x 154 mm x 138 mm) with paddy husk bedding. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited.
Enrichment
Tunnels were provided for individually housed animals.
Diet
Teklad Certified Global 14% Protein Rodent Maintenance Diet (Lot Number: 2014C-091719MA) from ENVIGO was provided ad libitum. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited.
Water
UV purified water was provided ad libitum. Results of bacteriological, chemical and contaminant analyses will be archived at RCC Laboratories India Private Limited.
Study design: in vivo (LLNA)
- Vehicle:
- dimethyl sulphoxide
- Details on study design:
- Dose formulation
The dose levels are in terms of the test substance as supplied. The dose formulations were prepared shortly before each dosing. The test substance was formulated in vehicle. The quantity of test substance was mixed with vehicle and the final volume was made up to 1000 µl for each group in a separate micro centrifuge tube. Homogeneity of the test substance in the vehicle was maintained during administration using a vortex mixer.
Note: DMSO (Dimethyl sulphoxide) was used as a vehicle for treatment group (Test substance) whereas acetone+ Olive Oil (4:1) were used as a vehicle for positive control group (Eugenol).
Selection of test substance concentrations for main study
A pretest was conducted to find out the appropriate test substance concentrations for main study. Two Female mice were used for each dose concentration. The dose concentrations used in the pretest are 10%, 25%, 50% and 100%. No mortality was observed at the concentrations of 10%, 25%, 50% and 100%. No clinical signs were observed at the concentrations of 10%, 25%, 50% and 100%. Animals were observed for body weight on Day 1 (prior to test substance application) and Day 6. Ears were observed for erythema on Day 1-6. Ear thickness was measured on Day 1 (pre dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Ear thickness and body weights were well within the normal range and no erythema was observed at the tested concentrations. The doses for the main study was selected based on the results of pretest and were described in an amendment to study plan. Pretest was performed before study initiation date and hence, excluded from statement of GLP. The doses selected for the main study was 10%, 25%, 50% and 100% for low, low mid, high mid and high doses, respectively. Considering the mortality, clinical signs and solubility of the test substance in the vehicle, the highest non-toxic concentration (100%) was selected for the main study.
Treatment
On Day 1, 2 and 3, the formulated test substance was applied to the dorsum of both the ears in a volume of 25 µL/ear using micropipette.
Justification: Dermal application is considered to be an appropriate application method as it is a possible route of human exposure during manufacture, handling and use of the test substance.
Dosing with BrdU
BrdU preparation
200 mg of BrdU was weighed into a tarred glass beaker on a weighing balance. 3 mL of phosphate buffer saline was added to beaker containing BrdU and mixed properly by using glass rod. This was then transferred to a 20 mL volumetric flask. Sufficient quantity of phosphate buffer saline was added to make up the required volume of solution (WN) to 20 mL. This was then transferred back to the beaker and mixed properly. The 10 mg/mL PBS solution containing BrdU was administered intraperitoneally to all the animals at a dose level of 5 mg/animal (0.5 mL/animal) on day 5. The BrdU solution was prepared freshly before administration.
Observation
MortalityNiability
Twice daily during the acclimatization period and during treatment and observation days, once on day of receipt and sacrifice.
Body Weights
On test days l (prior to test substance application) and 6 (Necropsy).
Clinical Signs
Once daily during the acclimatization period, at least twice daily approximately at 1st and 4th hours after test substance administration and on test day 1- 3, day 5 and once on day 4 and day 6.
Skin Reaction (Both Ears) Ear thickness
The skin reaction was assessed on days 1- 6 according to the numerical scoring system. Ear thickness was measured on Day I (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
Pathology
Necropsy
All the treated animals were sacrificed at the end of the observation period by carbon dioxide asphyxiation in euthanasia chamber. Gross/macroscopic pathological changes were observed and recorded. The auricular lymph nodes were excised from both ears of all the animals. The auricular lymph nodes were weighed and stored in ice-chilled Phosphate Buffer Saline (PBS) and kept in deep freezer (-20·C) until further processing. No organ and tissues were retained
6BrdU: ELISA
The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit (Roche Diagnostics). The assay procedure was as follows:
Lymph Node Cell (LNC) suspension Preparation
• The collected auricular lymph nodes were crushed using small pestle.
• 0.3 ml of PBS (Phosphate Buffer Saline) was added and the suspension was collected in a centrifuge tube.
• This suspension was pass through a nylon mesh (70µm). The volume of cell suspension was made up to 15 ml with PBS.
Assay Flow (BrdU- ELISA)
1. LNC suspension was centrifuged at 2000 rpm for ten minutes
2. ¾ th of supernatant solution was removed.
3. The cell suspension (100 µL) was added to Micro well plate (3 replicates /sample) and thoroughly mixed. This micro well plate was dried completely in a hot air oven at 45°C.
4. 200 µL of Fix-Denat solution was added to each well and allowed to stand for 30 minutes.
5. Fix-Denat solution was removed completely after 30 minutes.
6. 100 µL of anti-BrdU-POD antibody working solution was added and allowed to react for 1 hour.
7. Anti-BrdU-POD antibody working solution was removed completely after 1 hour.
8. 200 µL of wash solution (PBS) was added and washed the wells by pipetting 10 times. The solution was completely removed and discarded. Same step was repeated twice.
9. 100 µL of TMB (Tetra methyl Benzidine) substrate solution was added and allowed to stand for 15 minutes at room temperature in a dark place.
10. The absorbance was measured at 370 nm using spectramax reader.
Calculation
Stimulation Index (SI) was calculated as follows:
OD values for each test animal/ mean OD values for concurrent Negative control group
Accetability of the assay
The positive control response at an exposure level expected to give an increase in the SI greater than or equal to 1.6 over the negative control.
The SI >14 should be considered to be an excessive response. Induction at this level is considered to be not reproducible.
The SI 2:: 1.6 value of test substance is considered to be a positive response and the test substance a potential skin sensitizer.
Data compilation
Mortality/Viability, Clinical Signs, Skin Reactions, Ear Thickness measurements, Body Weights and Organ Weights were recorded on data sheets. All data generated by the 96-well plate ELISA reader was printed after reading and considered as raw data and excel sheet were used to calculate the Stimulation index. Data from individual animal was reported in tabular form. The Data was reported as mean± SD (standard deviation). - Positive control substance(s):
- eugenol (CAS No 97-53-0)
Results and discussion
In vivo (LLNA)
Resultsopen allclose all
- Key result
- Parameter:
- SI
- Value:
- ca. 1.07
- Test group / Remarks:
- 10% test substance
- Key result
- Parameter:
- SI
- Value:
- ca. 1.111
- Test group / Remarks:
- 25% test substance
- Key result
- Parameter:
- SI
- Value:
- ca. 1.119
- Test group / Remarks:
- 50% test substance
- Key result
- Parameter:
- SI
- Value:
- ca. 3.954
- Test group / Remarks:
- Positive control
- Key result
- Parameter:
- SI
- Value:
- ca. 1.154
- Test group / Remarks:
- 100% test substance
- Cellular proliferation data / Observations:
- Mortality
No mortality was observed in any of the animals treated during experimental period.
Clinical signs
All the animals appeared normal throughout the acclimatization period. All the animals appeared normal throughout the study observation period.
Body weights
The body weight of the animals was within the normal range of variability commonly recorded for this species, strain and age.
Skin reaction
The test substance did not induce any degree of erythema on the both ears of all animals in control and treatment groups on Day 1 to Day 6. Animals in positive control group showed well defined erythema on both the ears on Day 1 to Day 6.
Ear thickness
Ear thickness measurements in treated group animals were comparable with negative control group. Whereas ear thickness was slightly increased in positive control group compared with negative control group.
Macroscopic findings
No abnormalities were observed externally and internally in negative control, low, low mid, high mid and high dose group animals at necropsy. Externally both ears (Bilateral) found reddened, internally the size of the auricular lymph nodes (Bilateral) was enlarged slightly in positive control group at terminal sacrifice.
Organ weight
The size of the auricular lymph nodes was enlarged slightly in positive control group compared to negative control group.
BrdU ELISA results
A BrdU ELISA measurement with cell suspension of Auricular lymph nodes resulted in mean stimulation indexes of 1.070, 1.111, 1.119 and 1.154 at concentrations of 10, 25, 50 and 100%, respectively. Stimulation Index (SI) value of positive control was 3.954 and thus confirmed the validity of the test system.
Conclusion
The Stimulation Index (SI) of animals treated with test substance at concentrations of 10, 25, 50 and 100% was less than 1.6. Hence, the test substance is not a potential skin sensitizer.
Any other information on results incl. tables
Results
Table 1: Mortality, viability and clinical signs
Group |
Dose |
Animal Number |
Sex |
Test day |
|||||||||
1 |
2 |
3 |
4 |
5 |
6 |
||||||||
1 |
0%(NC) |
01 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
02 |
Female |
1 |
1 |
l |
1 |
1 |
l |
1 |
1 |
1 |
1 |
||
03 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
l |
1 |
1 |
1 |
||
04 |
Female |
l |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
05 |
Female |
1 |
1 |
1 |
I |
1 |
1 |
l |
1 |
1 |
1 |
||
2 |
10%(L) |
06 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
07 |
Female |
1 |
1 |
I |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
08 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
I |
1 |
1 |
l |
||
09 |
Female |
1 |
1 |
1 |
1 |
1 |
l |
1 |
1 |
1 |
1 |
||
10 |
Female |
1 |
1 |
I |
I |
1 |
1 |
1 |
1 |
I |
1 |
||
3 |
25%(LM) |
11 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
12 |
Female |
1 |
1 |
1 |
1 |
1 |
l |
1 |
1 |
1 |
1 |
||
13 |
Female |
1 |
l |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
14 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
15 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
4 |
50% (HM) |
16 |
Female |
l |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
17 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
l |
1 |
1 |
l |
||
18 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
19 |
Female |
1 |
1 |
1 |
1 |
1 |
l |
1 |
1 |
l |
1 |
||
20 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
5 |
100% (H) |
21 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
I |
22 |
Female |
1 |
1 |
I |
1 |
1 |
1 |
1 |
1 |
1 |
l |
||
23 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
24 |
Female |
I |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
25 |
Female |
1 |
1 |
I |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
6 |
25%(PC) |
26 |
Female |
1 |
1 |
1 |
1 |
I |
1 |
1 |
1 |
1 |
1 |
27 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
28 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
29 |
Female |
1 |
1 |
1 |
1 |
1 |
l |
1 |
l |
1 |
l |
||
30 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
Key: %=percentage, l= Normal, NC-Negative Control, L-Low, LM-Low Mid, HM- High Mid, H-High, PC-Positive Control
No clinical signs were evident in any animal during the acclimatization period.
Table 2: Body weight
Group |
Dose |
Animal Number |
Sex |
Test day 1 (pre-treatment) (g) |
Test day 6 (g) |
1 |
0% (NC) |
01 |
Female |
24.52 |
25.11 |
02 |
Female |
22.73 |
23.85 |
||
03 |
Female |
23.10 |
24.08 |
||
04 |
Female |
21.95 |
23.29 |
||
05 |
Female |
22.18 |
23.27 |
||
2 |
10% (L) |
06 |
Female |
23.78 |
25.75 |
07 |
Female |
22.69 |
24.41 |
||
08 |
Female |
22.50 |
23.41 |
||
09 |
Female |
21.83 |
23.70 |
||
10 |
Female |
22.13 |
23.89 |
||
3 |
25% (LM) |
11 |
Female |
23.62 |
24.70 |
12 |
Female |
22.75 |
23.43 |
||
13 |
Female |
22.43 |
23.25 |
||
14 |
Female |
21.76 |
23.84 |
||
15 |
Female |
21.91 |
23.33 |
||
4 |
50% (HM) |
16 |
Female |
23.29 |
23.96 |
17 |
Female |
22.80 |
24.37 |
||
18 |
Female |
22.69 |
24.33 |
||
19 |
Female |
22.15 |
23.52 |
||
20 |
Female |
21.90 |
22.84 |
||
5 |
100% (H) |
21 |
Female |
23.24 |
23.93 |
22 |
Female |
22.86 |
24.04 |
||
23 |
Female |
22.40 |
23.74 |
||
24 |
Female |
22.19 |
24.02 |
||
25 |
Female |
21.95 |
23.44 |
||
6 |
25% (PC) |
26 |
Female |
23.16 |
24.43 |
27 |
Female |
22.85 |
23.80 |
||
28 |
Female |
22.22 |
23.46 |
||
29 |
Female |
22.30 |
23.72 |
||
30 |
Female |
21.72 |
22.95 |
Key: %= percentage, g=gram, NC-Negative Control, L-Low, LM-Low Mid, HM- High Mid, H-High, PC-Positive Control
Table 3: Skin reactions
Reaction Scores |
||||||||||
Group |
Dose |
Animal Number |
Sex |
Site ofApplication |
Observation Day |
|||||
1 |
2 |
3 |
4 |
5 |
6 |
|||||
1 |
0% (NC) |
01 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
02 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
03 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
04 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
05 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
2 |
10% (L) |
06 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
07 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
08 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
09 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
10 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
Key: NC-Negative Control, L-Low.
Reaction Scores |
||||||||||
Group |
Dose |
AnimalNumber |
Sex |
Site ofApplication |
Observation Day |
|||||
1 |
2 |
3 |
4 |
5 |
6 |
|||||
3 |
25% (LM) |
11 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
12 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
13 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
14 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
15 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
4 |
50% (HM) |
16 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
17 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
18 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
19 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
20 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
Key: LM-Low Mid, HM-High Mid
Reaction Scores |
||||||||||
Group |
Dose |
Animal Number |
Sex |
Site of Application |
Observation Day |
|||||
1 |
2 |
3 |
4 |
5 |
6 |
|||||
5 |
100% (H) |
21 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
22 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
23 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
24 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
25 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
6 |
25% (PC) |
26 |
Female |
Right Ear |
0 |
2 |
2 |
2 |
2 |
2 |
Left Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||||
27 |
Female |
Right Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||
Left Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||||
28 |
Female |
Right Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||
Left Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||||
29 |
Female |
Right Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||
Left Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||||
30 |
Female |
Right Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||
Left Ear |
0 |
2 |
2 |
2 |
2 |
2 |
||||
Key: H-High; PC-Positive Control
Table 4: Ear thickness on Day 1
Group |
Dose |
Animal Number |
Sex |
Site of Application |
Ear Thickness (mm) |
Mean |
Mean±SD |
1 |
0% (NC) |
01 |
Female |
Right Ear |
0.280 |
0.279 |
0.280±0.0098 |
Left Ear |
0.278 |
||||||
02 |
Female |
Right Ear |
0.297 |
0.296 |
|||
Left Ear |
0.294 |
||||||
03 |
Female |
Right Ear |
0.272 |
0.271 |
|||
Left Ear |
0.269 |
||||||
04 |
Female |
Right Ear |
0.274 |
0.273 |
|||
Left Ear |
0.272 |
||||||
05 |
Female |
Right Ear |
0.283 |
0.282 |
|||
Left Ear |
0.281 |
||||||
2 |
10% (L) |
06 |
Female |
Right Ear |
0.286 |
0.285 |
0.285±0.0061 |
Left Ear |
0.284 |
||||||
07 |
Female |
Right Ear |
0.291 |
0.290 |
|||
Left Ear |
0.289 |
||||||
08 |
Female |
Right Ear |
0.295 |
0.294 |
|||
Left Ear |
0.292 |
||||||
09 |
Female |
Right Ear |
0.288 |
0.287 |
|||
Left Ear |
0.286 |
||||||
10 |
Female |
Right Ear |
0.302 |
0.301 |
|||
Left Ear |
0.299 |
Key: mm-Millimeter; NC-Negative Control, L-Low
Group |
Dose |
AnimalNumber |
Sex |
Site of Aoolication |
Ear Thickness (mm) |
Mean |
Mean±SD |
3 |
25%(LM) |
11 |
Female |
Right Ear |
0.294 |
0.293 |
0.300±0.0084 |
Left Ear |
0.291 |
||||||
12 |
Female |
Right Ear |
0.308 |
0.307 |
|||
Left Ear |
0.306 |
||||||
13 |
Female |
Right Ear |
0.312 |
0.311 |
|||
Left Ear |
0.309 |
||||||
14 |
Female |
Right Ear |
0.292 |
0.291 |
|||
Left Ear |
0.289 |
||||||
15 |
Female |
Right Ear |
0.301 |
0.300 |
|||
Left Ear |
0.298 |
||||||
4 |
50% (HM) |
16 |
Female |
Right Ear |
0.298 |
0.297 |
0.298±0.0075 |
Left Ear |
0.295 |
||||||
17 |
Female |
Right Ear |
0.309 |
0.308 |
|||
Left Ear |
0.307 |
||||||
18 |
Female |
Right Ear |
0.303 |
0.302 |
|||
Left Ear |
0.301 |
||||||
19 |
Female |
Right Ear |
0.289 |
0.288 |
|||
Left Ear |
0.287 |
||||||
20 |
Female |
Right Ear |
0.297 |
0.296 |
|||
Left Ear |
0.294 |
Key: mm-Millimeter, LM-Low Mid, HM-High Mid
Group |
Dose |
AnimalNumber |
Sex |
Site ofApplication |
Ear Thickness (mm) |
Mean |
Mean±SD |
5 |
100% (H) |
21 |
Female |
Right Ear |
0.314 |
0.313 |
0.304±0.0062 |
Left Ear |
0.311 |
||||||
22 |
Female |
Right Ear |
0.299 |
0.298 |
|||
Left Ear |
0.297 |
||||||
23 |
Female |
Right Ear |
0.308 |
0.307 |
|||
Left Ear |
0.305 |
||||||
24 |
Female |
Right Ear |
0.299 |
0.298 |
|||
Left Ear |
0.297 |
||||||
25 |
Female |
Right Ear |
0.307 |
0.306 |
|||
Left Ear |
0.304 |
||||||
6 |
25% (PC) |
26 |
Female |
Right Ear |
0.294 |
0.293 |
0.299±0.0100 |
Left Ear |
0.292 |
||||||
27 |
Female |
Right Ear |
0.315 |
0.314 |
|||
Left Ear |
0.313 |
||||||
28 |
Female |
Right Ear |
0.304 |
0.303 |
|||
Left Ear |
0.301 |
||||||
29 |
Female |
Right Ear |
0.289 |
0.288 |
|||
Left Ear |
0.287 |
||||||
30 |
Female |
Right Ear |
0.298 |
0.297 |
|||
Left Ear |
0.296 |
Key: mm-Millimeter, H-High, PC-Positive Control
Table 5: Ear thickness on Day 3
Group |
Dose |
Animal Number |
Sex |
Site ofApplication |
Ear Thickness (mm) |
Mean |
Mean±SD |
I |
0% (NC) |
01 |
Female |
Right Ear |
0.282 |
0.281 |
0.282 ± 0.0095 |
Left Ear |
0.279 |
||||||
02 |
Female |
Right Ear |
0.299 |
0.298 |
|||
Left Ear |
0.296 |
||||||
03 |
Female |
Right Ear |
0.275 |
0.274 |
|||
Left Ear |
0.273 |
||||||
04 |
Female |
Right Ear |
0.276 |
0.275 |
|||
Left Ear |
0.274 |
||||||
05 |
Female |
Right Ear |
0.286 |
0.285 |
|||
Left Ear |
0.284 |
||||||
2 |
10% (L) |
06 |
Female |
Right Ear |
0.288 |
0.287 |
0.287 ± 0.0061 |
Left Ear |
0.286 |
||||||
07 |
Female |
Right Ear |
0.294 |
0.293 |
|||
Left Ear |
0.292 |
||||||
08 |
Female |
Right Ear |
0.297 |
0.296 |
|||
Left Ear |
0.294 |
||||||
09 |
Female |
Right Ear |
0.291 |
0.290 |
|||
Left Ear |
0.289 |
||||||
10 |
Female |
Right Ear |
0.304 |
0.303 |
|||
Left Ear |
0.302 |
Key: mm-Millimeter; NC-Negative Control, L-Low
Group |
Dose |
AnimalNumber |
Sex |
Site of Aoolication |
Ear Thickness (mm) |
Mean |
Mean±SD |
3 |
25% (LM) |
11 |
Female |
Right Ear |
0.296 |
0.295 |
0.303± 0.0081 |
Left Ear |
0.294 |
||||||
12 |
Female |
Right Ear |
0.311 |
0.310 |
|||
Left Ear |
0.308 |
||||||
13 |
Female |
Right Ear |
0.314 |
0.313 |
|||
Left Ear |
0.311 |
||||||
14 |
Female |
Right Ear |
0.295 |
0.294 |
|||
Left Ear |
0.292 |
||||||
15 |
Female |
Right Ear |
0.304 |
0.303 |
|||
Left Ear |
0.301 |
||||||
4 |
50% (HM) |
16 |
Female |
Right Ear |
0.301 |
0.300 |
0.301± 0.0074 |
Left Ear |
0.298 |
||||||
17 |
Female |
Right Ear |
0.312 |
0.311 |
|||
Left Ear |
0.309 |
||||||
18 |
Female |
Right Ear |
0.305 |
0.304 |
|||
Left Ear |
0.303 |
||||||
19 |
Female |
Right Ear |
0.291 |
0.291 |
|||
Left Ear |
0.290 |
||||||
20 |
Female |
Right Ear |
0.299 |
0.298 |
|||
Left Ear |
0.297 |
Key: mm-Millimeter, LM-Low Mid, HM-High Mid
Group |
Dose |
Animal Number |
Sex |
Site of Application |
Ear Thickness (mm) |
Mean |
Mean±SD |
5 |
100% (H) |
21 |
Female |
Right Ear |
0.317 |
0.316 |
0.307 ± 0.0064 |
Left Ear |
0.314 |
||||||
22 |
Female |
Right Ear |
0.302 |
0.301 |
|||
Left Ear |
0.300 |
||||||
23 |
Female |
Right Ear |
0.311 |
0.310 |
|||
Left Ear |
0.308 |
||||||
24 |
Female |
Right Ear |
0.302 |
0.301 |
|||
Left Ear |
0.299 |
||||||
25 |
Female |
Right Ear |
0.311 |
0.310 |
|||
Left Ear |
0.308 |
||||||
6 |
25% (PC) |
26 |
Female |
Right Ear |
0.552 |
0.552 |
0.541± 0.0140 |
Left Ear |
0.551 |
||||||
27 |
Female |
Right Ear |
0.528 |
0.526 |
|||
Left Ear |
0.524 |
||||||
28 |
Female |
Right Ear |
0.561 |
0.560 |
|||
Left Ear |
0.558 |
||||||
29 |
Female |
Right Ear |
0.537 |
0.536 |
|||
Left Ear |
0.535 |
||||||
30 |
Female |
Right Ear |
0.533 |
0.532 |
|||
Left Ear |
0.531 |
Key: mm-Millimeter, H-High, PC-Positive Control
Table 6: Ear thickness on Day 6
Group |
Dose |
Animal Number |
Sex |
Site ofApplication |
Ear Thickness (mm) |
Mean |
Mean±SD |
1 |
0% (NC) |
01 |
Female |
Right Ear |
0.284 |
0.283 |
0.285±0.0093 |
Left Ear |
0.282 |
||||||
02 |
Female |
Right Ear |
0.300 |
0.300 |
|||
Left Ear |
0.299 |
||||||
03 |
Female |
Right Ear |
0.277 |
0.276 |
|||
Left Ear |
0.275 |
||||||
04 |
Female |
Right Ear |
0.279 |
0.278 |
|||
Left Ear |
0.277 |
||||||
05 |
Female |
Right Ear |
0.288 |
0.287 |
|||
Left Ear |
0.286 |
||||||
2 |
10% (L) |
06 |
Female |
Right Ear |
0.289 |
0.288 |
0.288±0.0064 |
Left Ear |
0.287 |
||||||
07 |
Female |
Right Ear |
0.297 |
0.296 |
|||
Left Ear |
0.295 |
||||||
08 |
Female |
Right Ear |
0.300 |
0.299 |
|||
Left Ear |
0.298 |
||||||
09 |
Female |
Right Ear |
0.294 |
0.293 |
|||
Left Ear |
0.292 |
||||||
10 |
Female |
Right Ear |
0.306 |
0.305 |
|||
Left Ear |
0.304 |
Key: mm-Millimeter; NC-Negative Control, L-Low
Group |
Dose |
Animal Number |
Sex |
Site ofApplication |
Ear Thickness (mm) |
Mean |
Mean±SD |
3 |
25% (LM) |
11 |
Female |
Right Ear |
0.299 |
0.298 |
0.306 ± 0.0081 |
Left Ear |
0.297 |
||||||
12 |
Female |
Right Ear |
0.314 |
0.313 |
|||
Left Ear |
0.311 |
||||||
13 |
Female |
Right Ear |
0.317 |
0.316 |
|||
Left Ear |
0.314 |
||||||
14 |
Female |
Right Ear |
0.298 |
0.297 |
|||
Left Ear |
0.295 |
||||||
15 |
Female |
Right Ear |
0.307 |
0.306 |
|||
Left Ear |
0.304 |
||||||
4 |
50% (HM) |
16 |
Female |
Right Ear |
0.304 |
0.303 |
0.303± 0.0075 |
Left Ear |
0.301 |
||||||
17 |
Female |
Right Ear |
0.314 |
0.313 |
|||
Left Ear |
0.312 |
||||||
18 |
Female |
Right Ear |
0.308 |
0.307 |
|||
Left Ear |
0.306 |
||||||
19 |
Female |
Right Ear |
0.294 |
0.293 |
|||
Left Ear |
0.292 |
||||||
20 |
Female |
Right Ear |
0.302 |
0.301 |
|||
Left Ear |
0.299 |
Key: mm-Millimeter, LM-Low Mid, HM-High Mid
Group |
Dose |
AnimalNumber |
Sex |
Site ofApplication |
Ear Thickness (mm) |
Mean |
Mean±SD |
5 |
100% (H) |
21 |
Female |
Right Ear |
0.319 |
0.318 |
0.310±0.0062 |
Left Ear |
0.316 |
||||||
22 |
Female |
Right Ear |
0.305 |
0.304 |
|||
Left Ear |
0.302 |
||||||
23 |
Female |
Right Ear |
0.314 |
0.313 |
|||
Left Ear |
0.311 |
||||||
24 |
Female |
Right Ear |
0.305 |
0.304 |
|||
Left Ear |
0.302 |
||||||
25 |
Female |
Right Ear |
0.314 |
0.313 |
|||
Left Ear |
0.311 |
||||||
6 |
25% (PC) |
26 |
Female |
Right Ear |
0.555 |
0.554 |
0.544±0.0140 |
Left Ear |
0.553 |
||||||
27 |
Female |
Right Ear |
0.531 |
0.530 |
|||
Left Ear |
0.528 |
||||||
28 |
Female |
Right Ear |
0.564 |
0.563 |
|||
Left Ear |
0.561 |
||||||
29 |
Female |
Right Ear |
0.541 |
0.540 |
|||
Left Ear |
0.539 |
||||||
30 |
Female |
Right Ear |
0.535 |
0.534 |
|||
Left Ear |
0.532 |
Key: mm-Millimeter, H-High, PC-Positive Control
Table 7: Macroscopic finding
Group |
Dose |
Animal Number |
Sex |
Mode of Death |
Macroscopic Finding |
|
External |
Internal |
|||||
I |
0% (NC) |
01 |
Female |
Terminal Sacrifice |
NAD |
NAD |
02 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
03 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
04 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
05 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
2 |
10% (L) |
06 |
Female |
Terminal Sacrifice |
NAD |
NAD |
07 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
08 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
09 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
10 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
3 |
25% (LM) |
11 |
Female |
Terminal Sacrifice |
NAD |
NAD |
12 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
13 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
14 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
15 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
4 |
50% (HM) |
16 |
Female |
Terminal Sacrifice |
NAD |
NAD |
17 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
18 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
19 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
20 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
Note:Dose expressed in concentration (%)
Key: NC-Negative control, L-Low, LM-Low Mid, HM-High Mid, NAD- No Abnormality Detected
Group |
Dose |
Animal Number |
Sex |
Mode of Death |
Macroscopic Finding |
|
External |
Internal |
|||||
5 |
100% (H) |
21 |
Female |
Terminal Sacrifice |
NAD |
NAD |
22 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
23 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
24 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
25 |
Female |
Terminal Sacrifice |
NAD |
NAD |
||
6 |
25% (PC) |
26 |
Female |
Terminal Sacrifice |
Ears (Bilateral)- Reddened, slightly |
Auricular lymph node (Bilateral)- enlarged,slightly |
27 |
Female |
Terminal Sacrifice |
Ears (Bilateral)- Reddened, slightly |
Auricularlymph node (Bilateral) - enlarged,slightly |
||
28 |
Female |
Terminal Sacrifice |
Ears (Bilateral)- Reddened, slightly |
Auricularlymph node (Bilateral)- enlarged,slightly |
||
29 |
Female |
Terminal Sacrifice |
Ears (Bilateral)- Reddened, slightly |
Auricularlymphnode (Bilateral) - enlarged,slightly |
||
30 |
Female |
Terminal Sacrifice |
Ears (Bilateral)- Reddened, slightly |
Auricularlymph node (Bilateral) - enlarged,slightly |
||
Note:Dose expressed in concentration (%)
Key: H- High, PC- Positive Control, NAD- No Abnormality Detected
Table 8: Organ weight
Group |
Dose |
Animal Number |
Sex |
Auricular Lymph nodes (gram) |
1 |
0% (NC) |
01 |
Female |
0.0069 |
02 |
Female |
0.0072 |
||
03 |
Female |
0.0064 |
||
04 |
Female |
0.0069 |
||
05 |
Female |
0.0054 |
||
2 |
10% (L) |
06 |
Female |
0.0061 |
07 |
Female |
0.0065 |
||
08 |
Female |
0.0076 |
||
09 |
Female |
0.0068 |
||
10 |
Female |
0.0059 |
||
3 |
25% (LM) |
11 |
Female |
0.0074 |
12 |
Female |
0.0063 |
||
13 |
Female |
0.0071 |
||
14 |
Female |
0.0068 |
||
15 |
Female |
0.0057 |
||
4 |
50% (HM) |
16 |
Female |
0.0059 |
17 |
Female |
0.0066 |
||
18 |
Female |
0.0069 |
||
19 |
Female |
0.0079 |
||
20 |
Female |
0.0082 |
||
5 |
100% (H) |
21 |
Female |
0.0068 |
22 |
Female |
0.0071 |
||
23 |
Female |
0.0083 |
||
24 |
Female |
0.0072 |
||
25 |
Female |
0.0069 |
||
6 |
25% (PC) |
26 |
Female |
0.0137 |
27 |
Female |
0.0124 |
||
28 |
Female |
0.0129 |
||
29 |
Female |
0.0141 |
||
30 |
Female |
0.0133 |
Key: NC-Negative control, L-Low, LM-Low Mid, HM- High Mid, H- High, PC- Positive Control
Table 9: BrDU ELISA reading
Group |
Dose |
Animal Number |
Sex |
OD Readings |
1 |
0% (NC) |
01 |
Female |
0.242 |
0.234 |
||||
0.224 |
||||
02 |
Female |
0.232 |
||
0.238 |
||||
0.245 |
||||
03 |
Female |
0.206 |
||
0.199 |
||||
0.201 |
||||
04 |
Female |
0.198 |
||
0.207 |
||||
0.210 |
||||
05 |
Female |
0.188 |
||
0.179 |
||||
0.191 |
||||
2 |
10% (L) |
06 |
Female |
0.236 |
0.229 |
||||
0.245 |
||||
07 |
Female |
0.228 |
||
0.224 |
||||
0.233 |
||||
08 |
Female |
0.258 |
||
0.241 |
||||
0.245 |
||||
09 |
Female |
0.217 |
||
0.208 |
||||
0.203 |
||||
10 |
Female |
0.216 |
||
0.211 |
||||
0.224 |
||||
3 |
25% (LM) |
11 |
Female |
0.261 |
0.253 |
||||
0.254 |
||||
12 |
Female |
0.247 |
||
0.236 |
||||
0.231 |
||||
13 |
Female |
0.228 |
||
0.232 |
||||
0.234 |
||||
14 |
Female |
0.237 |
||
0.243 |
||||
0.241 |
||||
15 |
Female |
0.223 |
||
0.214 |
||||
0.217 |
Key: NC-Negative Control, L- Low, Low Mid
Group |
Dose |
Animal Number |
Sex |
OD Readings |
4 |
50% (HM) |
16 |
Female |
0.242 |
0.236 |
||||
0.229 |
||||
17 |
Female |
0.224 |
||
0.219 |
||||
0.231 |
||||
18 |
Female |
0.252 |
||
0.259 |
||||
0.264 |
||||
19 |
Female |
0.233 |
||
0.226 |
||||
0.239 |
||||
20 |
Female |
0.229 |
||
0.248 |
||||
0.241 |
||||
5 |
100% (H) |
21 |
Female |
0.261 |
0.269 |
||||
0.254 |
||||
22 |
Female |
0.258 |
||
0.243 |
||||
0.247 |
||||
23 |
Female |
0.227 |
||
0.234 |
||||
0.236 |
||||
24 |
Female |
0.243 |
||
0.239 |
||||
0.248 |
||||
25 |
Female |
0.251 |
||
0.242 |
||||
0.239 |
Key: HM- High Mid, H-High
Group |
Dose |
Animal Number |
Sex |
OD Readings |
6 |
25% (PC) |
26 |
Female |
0.860 |
0.883 |
||||
0.878 |
||||
27 |
Female |
0.869 |
||
0.843 |
||||
0.830 |
||||
28 |
Female |
0.962 |
||
0.953 |
||||
0.909 |
||||
29 |
Female |
0.767 |
||
0.754 |
||||
0.757 |
||||
30 |
Female |
0.794 |
||
0.797 |
||||
0.778 |
Key: PC-Positive Control
Table 10: LLNA-BrDU ELISA mean readings
Animal No. |
Mean OD value |
AnimalNo. |
Mean OD value |
AnimalNo. |
Mean OD value |
AnimalNo. |
Mean OD value |
Gl |
G2 |
G3 |
G4 |
||||
01 |
0.233 |
06 |
0.237 |
11 |
0.256 |
16 |
0.236 |
02 |
0.238 |
07 |
0.228 |
12 |
0.238 |
17 |
0.225 |
03 |
0.202 |
08 |
0.248 |
13 |
0.231 |
18 |
0.258 |
04 |
0.205 |
09 |
0.209 |
14 |
0.240 |
19 |
0.233 |
05 |
0.186 |
10 |
0.217 |
15 |
0.218 |
20 |
0.239 |
Key: OD=OpticalDensity
Animal No. |
Mean OD value |
Animal No. |
Mean OD value |
GS |
G6 |
||
21 |
0.261 |
26 |
0.874 |
22 |
0.249 |
27 |
0.847 |
23 |
0.232 |
28 |
0.941 |
24 |
0.243 |
29 |
0.759 |
25 |
0.244 |
30 |
0.790 |
Key: OD=OpticalDensity
GI-Negative Control, G2-Low, G3-Low Mid, G4-High Mid, GS-High, G6-Positive Control
Table 11: Stimulation index
Group |
Animal No. |
SI value |
Mean±SD |
Minimum |
Maximum |
G2 |
06 |
1.113 |
1.070 ± 0.0729 |
0.981 |
1.164 |
07 |
1.071 |
||||
08 |
1.164 |
||||
09 |
0.981 |
||||
10 |
1.019 |
||||
G3 |
11 |
1.202 |
1.111 ± 0.0650 |
1.023 |
1.202 |
12 |
1.117 |
||||
13 |
1.085 |
||||
14 |
1.127 |
||||
15 |
1.023 |
||||
G4 |
16 |
1.108 |
1.119 ± 0.0575 |
1.056 |
1.211 |
17 |
1.056 |
||||
18 |
1.211 |
||||
19 |
1.094 |
||||
20 |
1.122 |
||||
GS |
21 |
1.225 |
1.154 ± 0.0494 |
1.089 |
1.225 |
22 |
1.169 |
||||
23 |
1.089 |
||||
24 |
1.141 |
||||
25 |
1.146 |
||||
G6 |
26 |
4.103 |
3.955 ± 0.3357 |
3.563 |
4.418 |
27 |
3.977 |
||||
28 |
4.418 |
||||
29 |
3.563 |
||||
30 |
3.709 |
Key: G2-Low, G3-Low Mid, G4-High Mid, GS-High, G6-Positive Control
Table 12: Pre-test
(1) Ear thickness on Day 1
Group |
Dose |
Animal Number |
Sex |
Site of Aoolication |
Ear Thickness (mm) |
Mean |
Mean±SD |
1 |
10% |
01 |
Female |
Right Ear |
0.298 |
0.298 |
0.301± 0.0053 |
Left Ear |
0.297 |
||||||
02 |
Right Ear |
0.306 |
0.305 |
||||
Left Ear |
0.304 |
||||||
2 |
25% |
03 |
Female |
Right Ear |
0.319 |
0.318 |
0.313± 0.0067 |
Left Ear |
0.316 |
||||||
04 |
Right Ear |
0.309 |
0.308 |
||||
Left Ear |
0.307 |
||||||
3 |
50% |
05 |
Female |
Right Ear |
0.305 |
0.306 |
0.313± 0.0095 |
Left Ear |
0.307 |
||||||
06 |
Right Ear |
0.321 |
0.320 |
||||
Left Ear |
0.318 |
||||||
4 |
100% |
07 |
Female |
Right Ear |
0.299 |
0.299 |
0.300 ±.0021 |
Left Ear |
0.298 |
||||||
08 |
Right Ear |
0.302 |
0.302 |
||||
Left Ear |
0.301 |
Key: mm-Millimeter
(2) Ear thickness on Day 3
Group |
Dose |
Animal Number |
Sex |
Site ofApplication |
Ear Thickness (mm) |
Mean |
Mean±SD |
1 |
10% |
01 |
Female |
Right Ear |
0.300 |
0.300 |
0.303 ± 0.0046 |
Left Ear |
0.299 |
||||||
02 |
Right Ear |
0.307 |
0.306 |
||||
Left Ear |
0.305 |
||||||
2 |
25% |
03 |
Female |
Right Ear |
0.321 |
0.320 |
0.315 ± 0.0071 |
Left Ear |
0.318 |
||||||
04 |
Right Ear |
0.311 |
0.310 |
||||
Left Ear |
0.308 |
||||||
3 |
50% |
05 |
Female |
Right Ear |
0.308 |
0.309 |
0.316 ± 0.0092 |
Left Ear |
0.310 |
||||||
06 |
Right Ear |
0.323 |
0.322 |
||||
Left Ear |
0.321 |
||||||
4 |
100% |
07 |
Female |
Right Ear |
0.302 |
0.302 |
0.303 ± 0.0014 |
Left Ear |
0.301 |
||||||
08 |
Right Ear |
0.304 |
0.304 |
||||
Left Ear |
0.303 |
Key: mm-Millimeter
(3) Ear thickness on Day 6
Group |
Dose |
Animal Number |
Sex |
Site ofApplication |
Ear Thickness (mm) |
Mean |
Mean±SD |
l |
10% |
01 |
Female |
Right Ear |
0.303 |
0.302 |
0.305 ± 0.0046 |
Left Ear |
0.300 |
||||||
02 |
Right Ear |
0.309 |
0.308 |
||||
Left Ear |
0.307 |
||||||
2 |
25% |
03 |
Female |
Right Ear |
0.323 |
0.322 |
0.317± 0.0067 |
Left Ear |
0.321 |
||||||
04 |
Right Ear |
0.314 |
0.313 |
||||
Left Ear |
0.311 |
||||||
3 |
50% |
05 |
Female |
Right Ear |
0.312 |
0.312 |
0.318±0.0014 |
Left Ear |
0.311 |
||||||
06 |
Right Ear |
0.326 |
0.325 |
||||
Left Ear |
0.324 |
||||||
4 |
100% |
07 |
Female |
Right Ear |
0.305 |
0.304 |
0.305 ± 0.0014 |
Left Ear |
0.303 |
||||||
08 |
Right Ear |
0.307 |
0.306 |
||||
Left Ear |
0.305 |
Key: mm-Millimeter
Table 14: Body weight
Group |
Dose |
Animal Number |
Sex |
Test Day 1 (pre-treatment) (g) |
Test Day 6(Necropsy)(g) |
1 |
10% |
01 |
Female |
20.74 |
22.28 |
02 |
Female |
21.16 |
23.18 |
||
2 |
25% |
03 |
Female |
20.10 |
21.32 |
04 |
Female |
20.59 |
21.75 |
||
3 |
50% |
05 |
Female |
19.95 |
21.42 |
06 |
Female |
21.23 |
22.85 |
||
4 |
100% |
07 |
Female |
20.16 |
21.92 |
08 |
Female |
20.64 |
22.53 |
Note: g-gram
Table 15: Clinical signs
Group |
Dose |
Animal Number |
Sex |
Test day |
||||||||
1 |
2 |
3 |
4 |
5 |
6 |
|||||||
I |
10% |
01 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
02 |
Female |
l |
1 |
I |
1 |
1 |
1 |
1 |
1 |
1 |
||
2 |
25% |
03 |
Female |
1 |
1 |
1 |
1 |
I |
l |
1 |
1 |
1 |
04 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
3 |
50% |
05 |
Female |
1 |
1 |
l |
1 |
1 |
1 |
1 |
1 |
1 |
06 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
4 |
100% |
07 |
Female |
1 |
l |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
08 |
Female |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
||
Key: l= Normal
Table 16: Dose foprmulation (pre-test)
Group |
Name |
Test Substance/Reference Substance Quantity |
Vehicle (DMSO) |
Final Concentration (%) |
1 |
Low dose |
100mg |
1000 µI |
10 |
2 |
Low mid |
250mg |
1000 µl |
25 |
3 |
High mid |
500mg |
1000 µI |
50 |
4 |
High dose |
1000 mg |
1000 µI |
100 |
Note: DMSO- Dimethyl Sulphoxide
Table 17: Irritation scoring
Group |
Dose |
AnimalNumber |
Sex |
Site ofApplication |
Observation day |
|||||
1 |
2 |
3 |
4 |
5 |
6 |
|||||
1 |
10% |
01 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
02 |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
|||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
2 |
25% |
03 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
04 |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
|||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
3 |
50% |
05 |
Female |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
06 |
Right Ear |
0 |
0 |
0 |
0 |
0 |
0 |
|||
Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
||||
4 |
100% |
07 |
Female |
Right Ear Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|||||
08 |
Right Ear Left Ear |
0 |
0 |
0 |
0 |
0 |
0 |
|||
0 |
0 |
0 |
0 |
0 |
0 |
|||||
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the study conditions, the test substance was not skin sensitizing.
- Executive summary:
A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 442B (Local Lymph Node Assay: BrdU -ELISA method), in compliance with GLP. Healthy animals were allocated to six groups, each consisting of five female BALB/c mice. Animals in Group 1 (GI) were applied with vehicle (DMSO) and served as negative control (NC). The test substance was applied to both ears at the concentrations of 10% (low), 25% (low mid), 50% (high mid) and 100% (high) for dose groups G2, G3, G4 and G5, respectively. In addition, animals in Group 6 (positive control) received the reference substance eugenol (25%) in acetone: olive oil (4:1). The dorsum of both ears was used as treatment site. The animals were observed for mortality at least twice daily during the acclimatization, treatment and observation days and at least once on day of receipt and sacrifice. Body weights were recorded on test Day 1 (prior to test substance administration) and Day 6. The animals were observed for clinical signs once daily during the acclimatization period, twice daily i.e., approximately at 1 and 4 h after test substance administration on test Days 1, 2, 3, 5 and once on Days 4 and 6. The skin reactions (both ears) and ear thickness were measured on Days 1 to 6 and Days 1, 3 and 6, respectively. All animals were necropsied and examined macroscopically at the end of the observation period. The auricular lymph nodes were collected from both ears and analyzed with BrdU ELISA. All animals appeared normal throughout the acclimatization and experimental periods. Bodyweight was within the normal range of variability commonly recorded for this species, strain and age. No erythema was observed prior to the administration period. The vehicle did not cause any erythema in the negative control group. The test substance also did not induce any erythema in the low, low mid, high mid and high dose groups from Days 1 to 6. In the positive control group, all the animals showed well defined erythema on both the ears from Days 2 to 6. Ear thickness in the treated group animals was comparable with that of the negative control group, whereas ear thickness was slightly increased in the positive control group. At necropsy, no abnormalities were observed externally and internally in the negative control, low, low mid, high mid and high dose group animals, whereas externally ears (bilateral) were found to be reddened slightly and internally the size of the auricular lymph nodes (bilateral) was slightly increased in the positive control group in gross pathology. BrdU ELISA measurement with the positive control cell suspension of auricular lymph nodes resulted in a Stimulation Index (SI) value of 3.954. As the SI was greater than 1.6, sensitivity and validity of the test system were confirmed. BrdU ELISA measurement with treated groups (G2 to G5) resulted in SI of 1.070 - 1.154. As SI were less than 1.6, the test substance was considered to have no sensitization potential in the test system. Under the study conditions, the test substance was not sensitising to mouse skin (Madhu, 2020).
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