Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-OCT-2005 - 25-JAN-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Remarks:
RCC Ltd, 4452 Itingen, Switzerland
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: black solid
- Analytical purity: > 99 %
- Storage condition of test material: room temperature (range of 20 ± 5°C), light protected.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaHsdRcc(SPF )
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Service, Füllinsdorf / Switzerland
- Age at study initiation: 8- 12 weeks (beginning of acclimatization )
- Weight at study initiation: 16 g - 24 g (ordered )
- Housing: Individual in Makrolon type-2 cages with standard softwood bedding.
- Diet: Pelleted standard Kliba 3433, batch no . 39/05 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum
- Water: Community tap water from Itingen, available 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 + 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 10% and 25% (based a non-GLP local toxicity pre-test, 25% was the highest technically applicable concentration in the chosen vehicle)


No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
• In a non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v; A00), ethanol/water (7/3, v/v) and N,N-dimethylformamide (DMF). The pre-test results showed that 25 % in acetone/olive oil (4/1, v/v) was the highest technically applicable concentration. Only lower concentrations suitable for application could be produced with the other vehicles.
• In a non-GLP local toxicity pre-test with the suitable vehicle A00, concentrations were determined in three single animals each treated with one of three different concentrations (5 %, 10 % and 25 %) in both ears on three consecutive days.
After each topical application, the residual test item was found at all dosing sites.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle acetone/olive oil (4/1, v/v) was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained until start of treatment using an appropriate homogenizer.
Three groups each of five female mice were treated daily with the test item at concentrations of 5%, 10% and 25% in acetone/olive oil (4/1, v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days (application volume: 25 µl). 25% was the highest technically applicable concentration in the vehicle. A control group of five mice was treated with the vehicle acetone/olive oil (4/1, v/v) only. 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 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 3H-methyl thymidine measured in a ß-scintillation counter.

- 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.

DETERMINATION OF EAR THICKNESS
On days 1, 2 and 3 (before application) as well as on day 6 (prior to necropsy), the thickness of both ear (left and right) of each animal was measured using a micrometer.
Any other observation (erythema, colouration, presence of residual test item, crust, etc .) was noted at each time point.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations of body weights and dpm values were calculated.
Dunnett-test was conducted for assessment of the difference significance between the test item groups and the negative control (vehicle) group.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
>= 0.8 - <= 1.2
Variability:
SD: 0.3 and 0.7
Test group / Remarks:
all test groups
Remarks on result:
other: see Remark
Remarks:
5 % (w/v) test item: 1.2 ± 0.3 (SD) 10 % (w/v) test item: 1.2 ± 0.7 (SD) 25 % (w/v) test item: 0.8 ± 0.3 (SD) No dose-response relationship was observed. Calculation of the EC 3 value was not performed because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 404 - <= 629
Variability:
SD: from 79 to 381
Test group / Remarks:
all tested groups
Remarks on result:
other: Control: 516 ± 79 (SD) dpm /node 5 % (w/v) test item: 604 ± 175 (SD) dpm / node 10 % (w/v) test item: 629 ± 381 (SD) dpm / node 25 % (w/v) test item: 404 ± 169 (SD) dpm / node

Any other information on results incl. tables

No biological relevant changes in ear thickness occurred. No deaths occurred during the study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. After the first topical application, the residual test item was found at both dosing sites in all mice of the test item groups, persisting for the remainder of the in-life phase of the study. The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Statistical Analysis* 
Group Test item concentration dpm/node M (SD) S.I.M (SD) t value Conclusion
CG 1 - 516 (79) - - -
TG 2 5 % (w/v) 604 (175) 1.2(0.3) 0.61 --
TG 3 10 % (w/v) 629 (381) 1.2(0.7) 0.78 --
TG 4 25 % (w/v) 404 (169) 0.8(0.3) 0.77 --

S.I. = Stimulation Index

* Dunnett-test (G = 4, N= 20, t= 2.59)

-- no significant difference at p </= 0.05 (two sides)

No dose-response relationship was observed. Calculation of the EC3 value was not performed because no test concentrations produced a Stimulation Index (S.I.) of 3 or higher.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study STIMULATION INDICES of 1 .2, 1 .2 and 0.8 were determined with the test item at concentrations of 5%, 10 % and 25 % in acetone/olive oil (4/1, v/v), respectively. Therefore, the test substance was found to be a non-sensitizer.
Executive summary:

In order to study a possible contact allergenic potential of the test article, a GLP compliant Local Lymph Node Assay according to the OECD guideline No. 429 was performed. Three groups each of five female mice were treated daily with the test item at concentrations of 5%, 10 % and 25 % in acetone/olive oil (4/1, v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically applicable concentration in the vehicle. A control group of five mice was treated with the vehicle acetone/olive oil (4/1, v/v) only. 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 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 3H-methyl thymidine measured in a ß-scintillation counter. All treated animals survived the scheduled study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. After the first topical application, the residual test item was found at both dosing sites in all mice of the test item groups (Groups 2-4), persisting for the remainder of the in-life phase of the study. In this study STIMULATION INDICES of 1 .2, 1 .2 and 0.8 were determined with the test item at concentrations of 5%, 10 % and 25 % in acetone/olive oil (4/1, v/v), respectively. Calculation of the EC 3 value was not performed because no test concentrations produced a STIMULATION INDEX (S .I .) of 3 or higher. Based on this result, the test article was found to be a non-sensitizer when tested up to the highest applicable concentration of 25 % in acetone/olive oil (4/1, v/v).