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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-09-01 to 2004-09-07
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed GLP study

Data source

Reference Type:
study report
Report date:

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Acetoacet-o-Anisidide TTR

In vivo test system

Test animals

other: CBA/CaOlaHsd
Details on test animals and environmental conditions:
Number of animals for the pre-test (non-GLP): 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) group: 1
Age: 8 - 12 weeks (beginning of acclimatization)
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum,
Environment: temperature: 22 + 3°C
relative humidity: approx. 30-80%
artificial light: 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

5.0, 10.0, 25.0 % (w/v)
No. of animals per dose:
Details on study design:
Administration and exposure: For determination of the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed on two mice with concentrations of 2.5, 5, 10, and 25 % (w/v). The top dose of the test item is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. Four female mice were treated with different concentrations of the test item and
vehicle alone by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Five hours after intravenous injection, the mice were sacrificed and 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.

Data and Observations: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). In addition to the sensitising reactions the following observations and data were recorded: mortality/viability once daily, body weights prior to the first application and prior to necropsy, clinical signs (local/systemic) daily from start to the termination of in-life phase.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
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)/(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 above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
Results of the Positive Control Study performed in April 2004

S.I of alpha-Hexylcinnamaldehyde

5 %: 1.9
10 %: 6.1
25 % : 11.5

EC3 = (a-c) [(3-d)/(b-d)] + c = 6.3% (w/v)

Results of the Positive Control GLP Study performed in October 2004

S.I of alpha-Hexylcinnamaldehyde

5 %: 2.0
10 %: 3.0
25 % : 4.9

EC3 = (a-c) [(3-d)/(b-d)] + c = 9.9 % (w/v)

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: 5.0 % (w/v): 0.94 10.0 % (w/v): 0.90 25.0 % (w/v): 1.16
other: disintegrations per minute (DPM)
Remarks on result:
other: 0 % (w/v): 3337 5.0 % (w/v): 3126 10.0 % (w/v): 2993 25.0 % (w/v): 3876

Any other information on results incl. tables

Calculation and Results of individual Data

Vehicle: DMF

 Test item concentration % (w/v)  Group  Measurement DPM       Calculation   Result
       DPM-BG a)  number of lymph nodes  DPM per lymph node b) S.I. 
 -  BG I  0.0  -  -  -  -
 -  BG II  0.0  -  -  -
 -  CG I  3336.7  3336.7  8  417.1  
 5.0  TG 2  3126.3  3126.3  8  390.8  0.94
 10.0  TG 3  2992.5  2992.5  8  374.1  0.90
 25.0  TG 4  3876.1  3876.1  8  484.5  1.16

BG = Background (1 ml 5% trichloroaceticacid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information
The test item Acetoacet-o-Anisidide TTR was found to be not a skin sensitiser in this assay.
Executive summary:

In the study the test item Acetoacet-o-Anisidide TTR dissolved in DMF was assessed for its possible contact allergenic potential in accordance with OECD test guideline 429, adopted 2002 -04 -24.

For this purpose a local lymph node assay was performed using test item concentrations of 5.0, 10.0, and 25.0 %.

The animals did not show any clinical signs during the course of the study and no cases of mortality observed.

In this study Stimulation Indices (S.I.) of 0.94, 0.90, 1.16 were determined with the test item at concentrations of 5.0, 10.0, and 25.0 % (w/v) in DMF.

The test item Acetoacet-o-Anisidide TTR was found to be not a skin sensitiser in this assay.