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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28.04. - 12.05.2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

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
Test material form:
solid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBNCaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
16 females (main study)
Age: 8-12 weeks (beginning of acclimatization)
Body weight: 16-24 g (ordered)
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Conditions: Standard Laboratory Conditions. Air-conditioned with target ranges for room temperature 22 ±. 3 °C, relative humidity 30 - 70 % and 10 - 15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.
Accomodation: Individual in Makroion type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
Diet: Pelleted standard Kliba 3433, available ad libitum.
Water: Community tap water, ad libitum.

Study design: in vivo (LLNA)

Vehicle:
other: Ethanol:water, 7:3 (v/v)
Concentration:
0 %(control group), 2.5%, 5%, 10%
No. of animals per dose:
4 females (nulliparous and non-pregnant)
Details on study design:
In this study the test item shows a better solubility in ethanol:water, 7:3 (v/v) than in acetone:olive oil, 4: 1 (v/v), which is normally used by official recommendation, so ethanol:water, 7:3 (v/v) was selected and used as the vehicle in the tests.
In a non-GLP conform pre-test in two mice, test item concentrations of 1 %, 2.5 %, 5 % and 10 % (w/v) were tested on one ear each. After 21 hours, no irritation effects were observed at these concentrations after a single application.
10 % (w/v) was the highest technically applicable concentration in the chosen vehicle.
The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitization properties in CBNCaOlaHsd mice. The validation- / positive control study was performed with ALPHA-HEXYLCINNAMALDEHYDE in acetone:olive oil, 4: 1 (viv) using CBNCaOlaHsd mice (RCC Study Number 852080) between 07-JAN-2004 and 23-JAN-2004.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a skin sensitizer and an EC3 value of 11. 7 % (w/v) was derived.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
5.6
Variability:
% (w/v)
Parameter:
SI
Value:
0
Test group / Remarks:
Group 1 (control group)
Parameter:
SI
Value:
1.3
Test group / Remarks:
Group 2, test item concentration 2.5% (w/v)
Parameter:
SI
Value:
2.5
Test group / Remarks:
Group 3, test item concentration 5% (w/v)
Parameter:
SI
Value:
6.4
Test group / Remarks:
Group 4, test item concentration 10% (w/v)

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study STIMULATION INDICES of 1.3, 2.5 and 6.4 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v), respectively, in ethanol:water, 7:3 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).
The test item A 130 was found to be a skin sensitizer and an EC3 value of 5.6 % (w/v) was derived.
Executive summary:

In order to study a possible contact allergenic potential of A 130, three groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 10 % was the highest technically applicable concentration in the vehicle. A control group of four mice was treated with the vehicle (ethanol:water, 7:3 (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 H-methyl thymidine measured in a ß-scintillation counter.

To avoid loss of the test item applied to the outer ear surface the test item was carefully dosed. To avoid missing of the test item from the ears a hair dryer was used to immediately dry the wet ears.

All treated animals survived the scheduled study period.

No clinical signs were observed.

 

 Test item concentration % (w/v)

 S.I.

 
 Group 2

2.5 

 1.3

 
 Group 3

 5*

 2.5*

 
 Group 4

10* 

 6.4*

 
 

 EC3=5.6% (w/v)

 

A dose-response relation was observed.

*This value was used in calculation of EC3.