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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th June 2020 to 23rd November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline M3 (R2). Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals.
Qualifier:
according to guideline
Guideline:
other: Final Rules of the Animal Welfare Act regulations (Code of Federal Regulations, Title 9)
Qualifier:
according to guideline
Guideline:
other: Public Health Service Policy on Humane Care and Use of Laboratory Animals (Office of Laboratory Animal Welfare, Current edition )
Qualifier:
according to guideline
Guideline:
other: Guide for the Care and Use of Laboratory Animals (National Research Council, Current edition )
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

1
Reference substance name:
N'-[(2S)-1,1,1-TRIFLUOROPROPAN-2-YL]BENZOHYDRAZIDE
Cas Number:
1453473-00-1
IUPAC Name:
N'-[(2S)-1,1,1-TRIFLUOROPROPAN-2-YL]BENZOHYDRAZIDE
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 8 to 11 weeks
- Weight at study initiation: 18.3 to 25 g
- Housing: Animals were individually housed during the main study phase. Enrichment was provided according to a Testing Facility SOP. The housing was equipped with an automatic watering valve as specified in the USDA Animal Welfare Act
- Diet: Block Lab Diet  (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum.
- Water: Tap water was available ad libitum to each animal via an automatic watering system or a water bottle. HydroGel ® was offered at receipt and as needed
- Acclimation period: 8 to 22 days

ENVIRONMENTAL CONDITIONS
- Temperature: Target temperatures of 68°F to 79°F
- Humidity: A target relative humidity of 30% to 70% was maintained.
- Photoperiod: A 12-hour light/12-hour dark cycle was maintained.
- IN-LIFE DATES: From: To: 9th June 2020 to 23rd November 2020

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
Lot No.: 0000223910
Concentration:
Phase 1 - Irritation Screen Phase
1, 5, 10, 25, and 50%.

Phase 2 - MLLNA main study phase
1, 5, 10, 25, and 35%
No. of animals per dose:
Phase 1: 2
Phase 2: 5
Details on study design:
Phase 1 (Groups 1 to 5): The test article and vehicle were administered once on Days 1, 2, and 3 at approximately the same time (±1 hour) via dermal application to each outer ear using a micropipette. Approximately 25 µL was dosed to each ear.

Phase 2 (Groups 11 to 20): The test article, positive control article and vehicle were administered once on Days 1, 2, and 3 at approximately the same time (±1 hour) via dermal application to each outer ear using a micropipette. Approximately 25 µL was dosed to each ear. On Day 6, the cell proliferation marker article was administered intravenously (IV) via the tail vein at a dose of 0.25 mL/animal (20 µCi).

General In-life Assessments – Main Study (Phase 1 and 2) Animals
Mortality/Cageside Observations
At least twice daily (morning and afternoon) beginning upon arrival through termination/release.
Detailed Clinical Observations
At receipt, Day-1 approximately 2 to 4 hours after dosing, thereafter once daily during the dosing phase. Animals were removed from the cage. Observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior.
Individual Body Weights
Phase 1: daily until necropsy.
Phase 2: Day 1 and prior to the 3H thymidine injection on Day 6

Phase 2 animals (Groups 11 to 20) surviving until scheduled euthanasia were euthanized by carbon dioxide inhalation. When possible, the animals were euthanized rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied throughout the day. At the terminal necropsy, 5 hours following the 3 H-methyl thymidine injection, the animals were euthanized. The auricular lymph nodes were removed for analysis from all Phase 2 animals (Groups 11 to 20).

The auricular lymph nodes were placed into a plastic container on wet ice containing 5 ml of cold phosphate buffered saline (PBS). A nylon cell strainer was placed into a plastic petri dish, the lymph node/PBS mixture was poured onto the cell strainer, and the original tube was retained. A 1 mL syringe plunger was used to gently press the cells through the cell strainer. The strainer and the end of the plunger were rinsed with 5 mL of cold PBS. The strainer was removed from the Petri dish and the contents of the petri dish were transferred back into the original retained tube. The petri dish was rinsed with 5 mL of cold PBS, and the rinse added to the retained tube. The sample was centrifuged at 2000 rpm under refrigerated conditions for 10 minutes to pellet the cells and the supernatant was decanted and discarded. The pellet was resuspended in approx 10 mL of PBS and centrifuged again. The supernatant was decanted and discarded. The pellet was resuspended in 1 mL of 5% (w/v) trichloroacetic acid (TCA) to pellet, mixed well with a pipette, transferred to 1.5 mL Eppendorf tubes, and stored for 12 to 18 hours under refrigerated conditions (approximately 4ºC). The sample was then centrifuged at controlled room temperature at 12,400 rpm for 1 minute. The supernatant was decanted and discarded. The pellet was resuspended in 1 mL of 5% (w/v) TCA. The sample was transferred to a 20 mL glass scintillation vial and weighed. The vials were rinsed with 3 mL of Ultima Gold, and the rinse added to the vial. An additional 4 mL of Ultima Gold was added to the vial and mixed well by vortexing.

All radioactive samples collected were counted by liquid scintillation counting (LSC) for at least 5 minutes or 100,000 counts. The raw data were expressed as disintegrations per minute (dpm)
per animal and background values were determined. Individual values that fell within 2 times the background value were assigned a value of 0 (zero). The mean dpm for each group was calculated, and the Sensitization Index (SI) calculated as the ratio of the unbiased estimate of the mean (back transformed) of the treated group against the control group.

SI = mean dpm of treated group / mean dpm of vehicle group

If the SI was greater than 3.0, then the test article was identified as a potential sensitizer.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Group Pair-Wise Comparison (General ANOVA)
If the control group had a sample size less than three, no inferential statistics were calculated. If a particular endpoint and/or parameter within a given collection interval had the same value across all experimental units, no inferential statistics were calculated. For endpoints and/or parameters where all groups with sample sizes of 3 or greater were included, the normality of the residuals and homogeneity of variances were tested to determine if the data were approximately normal or if a log transformation or rank transformation was required. Levene’s test was used to assess homogeneity of group variances and Shapiro-Wilk’s test was used to test the normality of the residuals. For the raw data, if Levene’s test was not significant (p≥0.01) and Shapiro-Wilk’s test was not significant (p≥0.01), then a normal distribution was used. If either the Levene’s test was significant (p<0.01) or Shapiro-Wilk’s test was significant (p<0.01), normality and homogeneity of variances were tested with a log transformation used on the data.
For the log transformed data, if Levene’s test was not significant (p≥0.01) and Shapiro-Wilk’s
test was not significant (p≥0.01), then a log normal distribution was used. If either the Levene’s
test was significant (p<0.01) or Shapiro-Wilk’s test was significant (p<0.01), then a rank
transformation was used on the data.
For raw or log transformed data, a one-way analysis of variance was used to test each endpoint
for the effects of treatment. If the treatment effect was significant (p<0.05), linear contrasts were constructed for a Dunnett’s pair-wise comparison of treatment groups.
For rank transformed data, a Kruskal-Wallis test was used to test each endpoint for the effects
of treatment. If the treatment effect was significant (p<0.05), a non-parametric Dunn’s pair-wise comparison test of each treatment group with the control group was performed.

Results and discussion

Positive control results:
The positive control group (35% HCA) resulted in a sensitization index (SI) of 14.0 which is indicative of a sensitizer (SI ≥3). In addition, the SI correlated well with the mean dpm values, which were significantly increased in the 35% HCA group relative to the vehicle group.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
8.8
Test group / Remarks:
1% test item
Remarks on result:
positive indication of skin sensitisation based on QSAR/QSPR prediction
Key result
Parameter:
SI
Value:
5.4
Test group / Remarks:
5% test item
Remarks on result:
positive indication of skin sensitisation based on QSAR/QSPR prediction
Key result
Parameter:
SI
Value:
6.6
Test group / Remarks:
10% test item
Remarks on result:
positive indication of skin sensitisation based on QSAR/QSPR prediction
Cellular proliferation data / Observations:
The Cell Proliferation Marker used is 3H]-Methyl Thymidine.
Phase 2 (Groups 11 to 20): On Day 6, the cell proliferation marker article was administered intravenously (IV) via the tail vein at a dose of 0.25mL/animal (20 µCi).

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
LSN3532787 formulations were determined to be sensitizers at a concentration of ≥1% in the Murine Local Lymph Node Assay.
Executive summary:

The objective of this study was to evaluate the potential of the test article, LSN3532787 to cause or elicit skin sensitization reactions (allergic contact dermatitis) via the Murine Local Lymph Node Assay (MLLNA) in CBA/J, mice. 


The test article (1%, 5%, 10%, 25% and 35%), positive control article and vehicle were administered once on Days 1, 2, and 3 at approximately the same time (±1 hour) via dermal application to each outer ear using a micropipette. Approximately 25 µL was dosed to each ear. On Day 6, the cell proliferation marker article was administered intravenously (IV) via the tail vein at a dose of 0.25 mL/animal (20 µCi).


Mortality, clinical observations and changes in body weight were observed. At the terminal necropsy, 5 hours following the 3H-methyl thymidine injection, the animals were euthanized. The auricular lymph nodes were removed and prepared for analysis by radioanalysis. The mean dpm for each group was calculated, and  the Sensitization Index (SI) calculated as the ratio of the unbiased estimate of the mean (back transformed) of the treated group against the control group. 


All main study animals in groups 16 to 20 survived to the scheduled necropsy.


There were no significant findings or signs of local irritation in either the Irritation Screen or in the Main Study Phase for animals administered the vehicle or the test article.
Red discoloration of the skin (cervical region), wet hair on both ears and the cervical region, and oily hair in the cervical region, was observed in animals administered 35% HCA on Days 1 through 6. 


There was no effect on body weight during the course of this study. 


The positive control group (35% HCA) resulted in a sensitization index (SI) of 14.0 which is indicative of a sensitizer (SI ≥3). In addition, the SI correlated well with the mean dpm values, which were significantly increased in the 35% HCA group relative to the vehicle group. When LSN3532787 formulations were compared to the vehicle the sensitization indices were 8.8, 5.4, and 6.6 for the formulations at 1%, 5%, and 10% for the test article groups, respectively. 


LSN3532787 formulations were determined to be sensitizers at a concentration of ≥1% in the Murine Local Lymph Node Assay.