Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14. - 27.09.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to guideline under GLP
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dihydro-C14-aldehyde
- Physical state: liquid
- Storage condition of test material: At room temperature (range of 20 -25 °C), light protected. The container was tightly closed.
Storage under nitrogen.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Service CH-4414 Fullinsdorf/ Switzerland
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 g - 24 g
- Housing: Individual in Makrolon type2 cages with standard softwood bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 -23 °C,
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10 %, 25 % and 50 % in acetone/olive oil (4/1, v/v)
application volume, 25 µl, was spread over the entire
dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Irritation: After a single application, no irritation effects were observed at the dosing sites applied up to
the concentration of 50 %. One day after the single topical application, a slight ear erythema
was noted at the local sites dosed at 100 % (undiluted).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, exposure to at least one concentration of the test item resulted in an
incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).
- Second, the data are compatible with a conventional dose response, although
allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
not specified
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship, and
the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)l(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold
increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the
co-ordinates of the two pair of data lying immediately below and above the S.I. value of 3 on
the local lymph node assay dose response plot.

Results and discussion

Positive control results:
Not relevant

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
Group 2: 10% test substance (w/v)
Remarks on result:
other: Control: 0; 10% (w/v): 1.8; 25% (w/v): 3.5; 50% (w/v): 6.5
Key result
Parameter:
SI
Value:
6.5
Test group / Remarks:
Group 4: 50% test substance (w/v)
Key result
Parameter:
SI
Value:
3.5
Test group / Remarks:
Group 3; 25% test substance (w/v)
Parameter:
EC3
Value:
20.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per Lymph node: Control: 608; 10% (w/v): 1070; 25% (w/v): 2100; 50% (w/v): 3957

Any other information on results incl. tables

No remarks

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The estimated concentration of the test item required to produce a SI of 3 is: EC3 = (a-c) [(3-d)l(b-d)] + c = 20.6 % (w/v). For Dihydro-C14-aldehyde a sensitising potential could be concluded.
Executive summary:

In order to study a possible contact allergenic potential of Dihydro-CICAldehyde, three

groups each of four female mice were treated daily with the test item at concentrations of

10 %, 25 % and 50 % in acetonelolive oil (4/1, v/v) by topical application to the dorsum of

each ear lobe (left and right) for three consecutive days. A control group of four mice was

treated with the vehicle acetonelolive oil (4/1, v/v) only. Five days after the first topical

a lication the mice were injected intravenously into a tail vein with radio-labelled thymidine

(J H)p- methyl thymidine). Approximately five hours after intravenous injection, the mice were

sacrificed, the draining auricular lymph nodes excised and pooled per group. 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 3HTdR-methyl

thymidine measured in a p-scintillation counter.

All treated animals survived the scheduled study period.

No clinical signs were observed in any animals of the control group or Group 2 (10 %). On

the third application day, a slight ear swelling was observed at both dosing sites in all mice of

Group 3 (25 %), persisting for a total of three days. On the second application day, a slight

to moderate ear swelling was observed at both dosing sites in all mice of Group 4 (50 %), in

addition, on the third application day, a slight ear erythema was also noted. The local signs

in all mice of Group 4 (50 %) persisted for the remainder of the in-life phase of the study.

The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table. The

estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3

value.