Registration Dossier

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-06-2014 to 29-09-2014
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(2S,3S)-2,3-Bis[(4-methylbenzoyl)oxy]butanedioic acid — methyl {(4S)-8-fluoro-2-[4-(3methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4yl}acetate — ethyl acetate (1:1:1)
EC Number:
Cas Number:
Molecular formula:
(2S,3S)-2,3-Bis[(4-methylbenzoyl)oxy]butanedioic acid — methyl {(4S)-8-fluoro-2-[4-(3methoxyphenyl)piperazin-1-yl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydroquinazolin-4yl}acetate — ethyl acetate (1:1:1)
Test material form:
Details on test material:
lot L--005457795-001 G001

In vivo test system

Test animals

Details on test animals and environmental conditions:
Female CBNJ mice were received from Jackson Laboratories (Bar Harbor, ME) on 01 Jul 2014. Following an acclimation period of at least five days, fifteen nulliparous, non-pregnant female mice were assigned to the test groups using a computer-based random number program.
The animals were born on 20 May 2014 (+3 days). The Day 1 body weight range for the study animals was 17.2 - 21.2 grams. The weight variation of the animals at study start did not exceed +20% of the mean body weight. The animals were observed prior to the study start to ensure that no skin lesions were present on the ears.
The animals were identified by cage notation and indelible tail marks) and housed one per cage in suspended wire-bottom cages. Bedding was placed beneath the cages and changed at least three times per week. Fresh PMI Rodent Chow (Diet #5001) and water were available ad libitum. The animal room, reserved exclusively for mice on acute tests, was temperature-controlled, had a 12-hour light/dark cycle, and was kept clean and vermin free. Temperature and humidity were continuously monitored using automatic recording devices.

Study design: in vivo (LLNA)

25% w/v
No. of animals per dose:
Details on study design:
- Compound solubility:
The test article was tested for solubility in Acetone:Olive Oil (4:1, AOO) and N,N-Dimethylformamide (DMF). The test article was not soluble at 25% (w/v) in AOO, but was soluble at 25% in DMF

-All animals in the study were observed once daily throughout the study for any clinical signs, either of local irritation at the application site or systemic toxicity, and for mortality.

- Ear thickness measurements:
Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application (approximately 48 hours after the first test article application), and on Day 6 before sacrifice (approximately 120 hours after the first dose and 72 hours after the third dose). Changes in ear thickness on Day 3 and Day 6 relative to Day 1 were expressed as a percent of the Day 1 pre-dose values. Ear thickness increases of 25°/o or more were considered biologically significant (based on the scientific literature and historical laboratory data) and deemed indicative of a greater than moderate local dermal irritation response.


- Name of test method:
Local Lymph node assay
- Criteria used to consider a positive response:
A test article is considered to have a sensitizing potential if treatment results in a three-fold or greater increase in the number of proliferating lymphocytes relative to that obtained for the Vehicle Control. Therefore, test articles that yield an SI> 3 are characterized as potential sensitizing substances

Groups of five CBA/J mice were treated by a topical application of a single test article concentration (25% (w/v)), Vehicle Control (DMF) or Positive Control (25% HCA) to the dorsum of each ear once daily for three consecutive days. The Vehicle Control and Positive Control groups were shared with another study (MB# 14-22747.26). The test substance was spread over the entire dorsal surface of the ear using a micropipette at 25 µI/ear. The test article dose was 25% (w/v).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
For each test group, the individual animal SI values along with the mean group SI and standard deviation were calculated, and ANOVA followed by the Students' t-Test was run to statistically compare the test article dose group to the Vehicle Control group.

Results and discussion

Positive control results:
25% HCA in DMF induced a mean SI of 5.4 standard deviation 1.1

In vivo (LLNA)

Key result
ca. 0.8
standard deviation 0.3
Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION - the totla number of proliferating lymphocytes was calculated by multiplying the total cell number by the percentage of the cells that incorportate BrdU. SI was calculated by divding the total number of proliferating lymphocytes for each animal by the mean number of proliferating lymphocytes of the vehicle control group.

EC3 CALCULATION - see table below

CLINICAL OBSERVATIONS: All animals survived the in-life phase of the study and were observed to be normal. Treatment with 25% w/v of the test article did not induce excessive dermal irritation. The mean % change in ear thickness was 5.3% between day 1 and 3. the ear thickness change for the positive control was 15.8%. The same changes were observed on day 6 compared to day 1.

BODY WEIGHTS: Body weight changes were normal

Any other information on results incl. tables

 Cell proliferation numbers and Stimulation index

 Treatment  Animal #  Total # cells in node  %BrdU+  Lymphocyte proliferation  SI
 DMF 4202000 0.91


  4127000  0.80  33016  1.1 
  4246750  0.67 28453  1.0 
  4671250  0.6  28028  0.9 
  2962250  0.73  21624  0.7 
  mean  4041850  0.74  29872  1.0 
  St dev  639641  0.12  6191  0.2 
25% HCA  8108750  2.65  214882  7.2 
 positive control  7  9612667 1.39  133616  4.5 
  11451250  1.49  170624  5.7 
  9444250  1.53  144497  4.8 
  10  9214250  1.63  150192  5.0 
   mean  9566233 1.74  162762  5.4* 
  st dev  1206076  0.52  32091  1.1 
25% test article   26  4161500  0.47 19559  0.7 
  27  3543250  0.5  17716  0.6 
  28  4763500  0.68  32392  1.1 
  29  3980667  0.4  15923  0.5 
  30  444750  7.93  35269  1.2 
  mean   3378733  2.0  24172  0.8
   st dev  1697642  3.32  8968  0.3

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Topical application of the test article at 25% w/v in DMF vehicle resulted in an SI value less than 3.0 (SI < 3.0). Therefore this test article is not a potential dermal sensitiser at 25% in DMF in the Screening Locla Lymph Node Assay.
Executive summary:

Objective: To screen for possible sensitising potential of topically applied test items. The flow cytometry-modified LLNA protocol on which this screen is based is designed to be an alternative assay for the Buehler Guinea Pig Sensitisation Assay defined in the NIH report 'The Murine Local Lymph Node Assay: A test method for assessing the Allergenic Contact Dermatitis Potential of Chemicals/Compounds NIH No. 99-4494, 1999 and the LLNA as defined in EPPA OPPTS 870.2600, Final Guideline (March 2003) and OECD Guideline for the Testing of Chemicals No. 429, revised July 2010. 

Method Synopsis: The test article was tested for solubility in Acetone:Olive Oil (4:1, AOO) and N,N-Dimethylformamide (DMF). The test article was not soluble at 25% (w/v) in AOO, but was soluble at 25% in DMF. One group of five healthy female CBA/J mice was treated with a 25% (w/v) concentration of test article by topical application to the dorsum of each ear once daily for three consecutive days. A Vehicle Control group of five mice was treated with DMF and another group was treated with the Positive Control (alpha-Hexylcinnamaldehyde, 85% DMF 25% HCA) in the exact same manner. The mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2'-deoxy-uridine (BrdU) five days following intial dose and approximately five hours prior to sacrifice. At sacrifice, the auricular lymph nodes were isolated, single-cell suspensions of lymph node cells (LNC) were generated by flow cytometry for BrdU incorporation and the total number of LNC. The amount of proliferating (#BrdU+) LNC was determined as a measure of the proliferative response of the local lymph node. The Simulation Index (SI) was calculated by dividing the proliferative response (#BrdU + LNC) of each test article treated animal by the mean proliferative response of the Vehicle Control group. The mean SI ± SD was calculated for each group from the individual animal data. Test article groups that yielded ≥3 were determined as sensitising substances. 

Summary: All animals survived the in-life phase of the study and were observed to be normal. Body weight changes were normal. Ear thickness measurements and individual animal obervations indicated that the test article treatment did not result in dermal irritation. The SI of the Positive Control, 25% HCA was 5.4. The SI for the test article at 25% (w/v) was 0.8.