Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 10 and 50% (w/v)
No. of animals per dose:
5

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Test item concentration S.I. Vehicle Control Group (acetone:olive oil (4+1 v/v)) 1.00 1% GR-50-0091 1.42 10% GR-50-0091 1.68 50% GR-50-0091 6.59
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Test item concentration Mean DPM per animal (2 lymph nodes) Vehicle Control Group (acetone:olive oil (4+1 v/v)) 661.8 1% GR-50-0091 938.6 10% GR-50-0091 1109.8 50% GR-50-0091 4363.0

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In order to study a possible contact allergenic potential of GR-50-0091, three groups each
of five female mice were treated once daily with the test item at a concentrations of 1, 10,
and 50% (w/v) in acetone:olive oil (4+1 v/v) by topical application to the dorsum of each
ear for three consecutive days. The highest concentration tested was the highest
concentration that could be achieved whilst avoiding systemic toxicity and excessive local
skin irritation as confirmed by two pre-experiments. Two further groups each of five mice
were treated with the vehicle only (acetone:olive oil (4+1 v/v)) or with the positive control
item at 25% (w/v). Five days after the first topical application the mice were injected
intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine).
Approximately five hours after intravenous injection, the mice were sacrificed, the draining
auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph
node cells were prepared from pooled lymph nodes, which were subsequently washed and
incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph
node cells was determined by the incorporation of 3H-methyl thymidine measured in a -
scintillation counter.
Cases of mortality were not observed during the course of the study. Animals treated with
the highest test item concentration showed an erythema of the ear skin (Score 1) on
application day 4 and 5. A statistically significant increase in ear weight was seen in the
groups treated with 50 and 10% test item concentration; however, as the observed
increase (18.7% in the mid dose and 17.3% in the high dose group, respectively) was well
below the threshold of 25% for excessive skin irritation, this was not considered as
biologically relevant. Furthermore, the measured ear thickness did not show a relevant
increase.
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test
concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared
with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated
concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 1.42, 1.68, and 6.59 were determined with the test item
at concentrations of 1, 10, and 50% (w/v) in acetone:olive oil (4+1 v/v). A clear dose
response was observed. A statistically significant increase in the mean DPM value was
observed in the group treated with 50% test item concentration in comparison to the value
of the vehicle control group. The EC3 value calculated was 20.8 %. The S.I. determined
for the positive control was 9.56, demonstrating the validity of the study.
The test item GR-50-0091 was found to be a skin sensitiser under the test conditions of
this study.