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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: acccording to guideline and GLP and additional discriminating measurements sensitizing and irritationg properties

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The modification refers to the measurement of cell proliferation by cell counting instead of radioactive labelling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune system
Principles of method if other than guideline:
Modified LLNA (IMDS = Integrated Model for the Differentiation of Skin Reactions): The modification refer to the measurement of cell proliferation by cell counting instead of radioactive labelling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immun system, as also recommended in the update of OECD TG 429.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Dye content: 41.2 %

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: 26-36 g
- Housing: during the study individually
- Diet ad libitum
- Water. ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0% (vehicle control), 2.5 %, 10 %, 41.2 %, and positve control 30 % alpha hexyl cinnamic aldehyde in DMF
No. of animals per dose:
6
Details on study design:
The test item in the formulation, the positive control in the formulation or the vehicle were applied epicutaneousely onto the dorsal part of both ears.of the animals. This treatment was repeated on three consecutive days. The volume administered was 25 µl/ear. The used concentrations were based on the experience with this test system and the properties of the test item.
The animals were anaestetized by inhalation of carbon dioxid and sacrificed one day after the last application. The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline
Investigations:
-weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell count of the subtancetreated lymp nodes by the vehicle treated ones
- ear swelling
- ear weight
-body weight
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group by one-way Analysis of Vartiance (ANOVA) when the variances are considered homogenous according to a homogenicity testig like Cochran's test. Alternatively, if the variances are considered to be heterogenous a non-parametric Kruskal-Wallis test has been used at significance levels of 5 %.. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99 % by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Sheffe's method, wich can be used for both equal and unequal sample sizes.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
It has to be clarified that the positive levels are exclusively defined for the NMRI mice used for this study: Compared to vehicle treated animals, none of the parameter measured in the test substance groups i.e. cell counts, weights of the draining lymph nodes, ear weights and ear swelling reached or exceeded the 'positive level' defined for this assay. The cell count indices were determined 1.0, 0.92, 0.97, 1.16. Thus, the positive level, which is 1.4 for cell count index was never reached or exceeded. The respective value for the positive controls is 1.61
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labelling.

Any other information on results incl. tables

It has to be clarified that the positive levels are exclusively difined for the NMRI mice used for this study:

The positive level of ear swelllling which is 2x10 [exp -2] mm increase , i.e. about 10 % of the control values, has not been reached or exceeded in any dose groups of the test item whereas the respective value of the positive control has shown a statistical significant increase.

body weights of the animals was not affected by any treatment.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of formulation containing 0%, 2.5 %, 10 %, or 41.2 % of the test item Bayscript Blaukomponenten in DMF for 3 consecutive days onto both ears of the animals. The study was conducted according to OECD TG No. 429 and No 406. Bayscript Blaukomponente showed no sensitizing potential in the modified Local Lymph Node Assay (IMDS) in female NMRI mice after dermal application of up to and including a 41.2 % concentration. Additionally, no indication for a non-specific(irritant) activation by the test item was detected. The positive control hexyl cinnamic aldehyde (CAS-No. 101 -86 -0) was functional for each parameter (Vohr 2012). According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.
Executive summary:

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of formulation containing 0%, 2.5 %, 10 %, or 41.2 % of the test item Bayscript Blaukomponenten in DMF for 3 consecutive days onto both ears of the animals. The study was conducted according to OECD TG No. 429 and No 406. Bayscript Blaukomponente showed no sensitizing potential in the modified Local Lymph Node Assay (IMDS) in female NMRI mice after dermal application of up to and including a 41.2 % concentration. Additionally, no indication for a non-specific(irritant) activation by the test item was detected. The positive control hexyl cinnamic aldehyde (CAS-No. 101 -86 -0) was functional for each parameter (Vohr 2012). According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.