Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to official guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: V516690
Appearance: Brown powder
Batch: V516690/AY
Purity/Composition: LC-MSpurity (Area% UV-VIS): 95.8% (Reference: PA064492)
Test item storage: At room temperature
Stable under storage conditions until 17 July 2016 (expiry date)
Molecular formula: C49H49ClN2O6S2
MW: 861.52

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species and strain: Mice, CBA/JN
Sex: females (nulliparous and non-pregnant)
Age: 7 weeks old, approx. 25 grams
Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy
Breeder: Charles River Italia S.p.A., Calco (Lecco), Italy
Weight range at arrival: 17.9 to 19.3 grams
Acclimatisation period: at least 5 days
Veterinary health check during the acclimatisation period


Housing: 1/cage during the study; up to 5 during acclimatisation Polysulphone solid bottomed cages measuring 35.5 × 23.5 × 19 cm with nesting material
Cage control: Daily inspected and changed as necessary (at least twice/week)
Water: drinking water supplied to each cage via a water bottle
Water supply: ad libitum
Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet suply: ad libitum throughout the study
Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hour
Air changes: Approximately 15 to 20 air changes per hour
Temperature range: 22 °C ± 2 °C
Relative humidity range: 55 % ± 15 %

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main assay: 0 (vehicle), 2.5, 5, 10 %w/w
positive control: 25 %w/w
No. of animals per dose:
4 animals per dose
Details on study design:
A preliminary test was carried out to select three concentrations to be used in a Main Assay, according to the criteria described in the relevant guideline for this test. A Main Assay was then carried out to fully evaluate lymph node cell reaction.
Animal identification was permanent, following arrival, by ink marking on the tail. Animals were identified by odd numbers. Animals were randomised at arrival. Healthy animals without observable skin lesions were chosen. At the time of treatment (for preliminary test and Main Assay) the animals aged approximately 8-9 weeks.
Euthanasia method: The animals were sacrificed on Day 6 by carbon dioxide narcosis.
Necropsy procedure: After sacrifice, regularly shaped biopsies were obtained from both ears and weighed together. No necropsy was performed on the animals.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Differences between each treated group and the concurrent negative control group (indi- vidual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.
Assay validity criteria: The assay is considered satisfactory if the Stimulation Index (SI) of the positive control group is higher than 2.0.

Results and discussion

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
An increase in cell proliferation of draining lymph nodes, statistically significant at the highest concentration, was observed in all dose groups, with a Stimulation Index of 2.21, 1.84 and 2.87 at the low, medium and high dose (2.5 %, 5 % and 10 %), respectively. The lack of dose-relation was ascribed to inhomogeneous values of absorbance within Group 3. An abnormally high value for animal no. 41 of Group 4 (Replicate 1) was also observed. Nevertheless, values of SI were above the threshold of 1.6 for all treated groups. In the group treated with the positive control item, a Stimulation Index of 9.30 was calculated. As it was greater than 2, the study was regarded as valid.

Any other information on results incl. tables

No mortality and no clinical signs were recorded in animals treated at all dose levels investigated [10, 5, and 2.5 % (w/w)].

Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Applicant's summary and conclusion

Conclusions:
The potential of the test item, V516690, to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b. An increase in cell proliferation of draining lymph nodes was observed in all treatment groups. The calculated Stimulation Indices were 2.21, 1.84 and 2.87 at low, mid- and high dose levels, respectively. European legislation concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would indicate the following: Classification : Category 1 ; Signal word: Warning ; Hazard statement: H317: May cause an allergic skin reaction