Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4.9. - 13.9.2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: different endpoint than OECD/EC test guidelines but well validated. GLP compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
other endpoint
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
other endpoint
Principles of method if other than guideline:
endpoint different; modifications as described in publications of Ulrich P, Streich J, Suter W, 2001; Ehling et aI.,
2005; Ehling et aI., 2005A.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl N-[(1S,3S,4S)-4-hydroxy-4-[4-methoxy-3-(3-methoxypropoxy)phenyl]-1-[(2S,4S)-5-oxo-4-(propan-2-yl)oxolan-2-yl]-3-(propan-2-yl)butyl]carbamate
EC Number:
700-317-0
Cas Number:
934841-33-5
Molecular formula:
C30H49NO8
IUPAC Name:
tert-butyl N-[(1S,3S,4S)-4-hydroxy-4-[4-methoxy-3-(3-methoxypropoxy)phenyl]-1-[(2S,4S)-5-oxo-4-(propan-2-yl)oxolan-2-yl]-3-(propan-2-yl)butyl]carbamate
Details on test material:
Test substance name: SPPIOO C6
Mol. weight:551.73
CAS No.: not available
Batch No.:HH-853.35Kr
Purity: 99.2 % (A) - HPLC (Sum of isomers 1 and 2)
Significant impurities:sum of all impurities by HPLC: ........ 0.8 % (A)
Solvent content:
Isopropyl acetate: (CAS No. 108-21-4) ....... 1.9 % (w/w)
Methylcyclohexane: (CAS No. 108-87-2) ... 5.8 % (w/w)
Stability/Expiration: DEC-2007
findings of test facility:
Appearance: white, fine-crystallic powder
pH: approximately 5 (universal indicator papers pH 0 - 12)


approximately 5 (universal indicator papers pH 0 - 12)

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
Animals species and strain: Young adult female mice (nulliparous and non-pregnant), strain
BALB/CBYJICO. Source: Breeding farm BioTest s.r.o., KOlllirovice, 281 25 Czech
Republic, RCH CZ 21760152. 8 to 10 weeks (at start of dosing) 18 to 22 g (at start of dosing

Room temperature 22 ± 3°C, permanently monitored
Relative humidity 30 - 70 %, permanently monitored
Light: 12 hours lightldark cycle: 6am-6pm/6pm-6am
Group of maximum six animals in macrolon cages with
sterilized softwood shavings
Drinking tap water ad libitum. Water quality corresponded to
Regulation No. 25212004 Czech ColI. of Law, Health Ministry
Pelleted standard diet for experimental animals ad libitum.
Microbiological control and content of nutrients will be
performed according to SOP No. 75 and 83
Cleaning and disinfection of animal room was regularly
performed as it is described in appropriate SOP No.10

Study design: in vivo (non-LLNA)

Positive control substance(s):
yes
Remarks:
0.5% (w/v) Dinitrochlorbenzene (DNCB)

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 - mixture of40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
0% (vehicle) 30, 3 and 0.3 % (w/v)
No. of animals per dose:
6
Details on study design:
Six mice per group were exposed by test and control substances on the dorsum of both ears (25 uL per ear) once a day during 3 consecutive days. Lymph nodes were taken 24 hours after the last application. Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and SPPIOO C6: 30%, 3%, 0.3% (w/v). Endpoints: ear weight, auricular (ear-draining) lymph node weights and cell counts = lymph node (LN)
hyperplasia.
Statistics:
Statgraphic ® Centurion (version XV, USA); non-parametric Kruskal-Wallis test, followed by non-parametric two-group Mann-Whitney rank test (probability level 0.05)

Results and discussion

Positive control results:
cell concentration in suspension and weight of lymph nodes was statistically significantly increased against negative control group, indicating that the LLNA test was efficient.

In vivo (LLNA)

Results
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable

Any other information on results incl. tables

In the positive control groups (with DNCB) the LN weight, LN cell count and ear weight were statistically significantly increased from negative control values. This result demonstrates that method performed in conditions of our laboratory has enough sensitivity to detect the endpoint in view. The test substance SPPlOO C6 exposed animals showed no pathological skin reactions and only slight negative clinical changes throughout the experiment. The increase of the body weight of animals throughout the study denoted no toxic affect ofthe treatment. The test substance SPP 1 00 C6 did not showed a tendency to increase ear weight at any tested dose level. Comparison of values between treated groups and control group revealed that the test substance SPP100 C6 did not cause statistically significant increase in LN cell count and LN weight in any dose levels. Also index of LN weight and LN cell count was not exceeded. According to the criteria the results were considered as negative.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the given test conditions, the test substance, SPPIOO C6, did not give positive result in LLNA test.