Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- modification: in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance (Integrated Model for the Differentiation of Skin reactions (IMDS))
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Strain: Hsd Win:NMRI
- Housing: in groups up to 6 animals
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): about 50
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
other: polyethylene glycol 400
Concentration:
0, 1, 10, 50 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d0, d1 and d2). The volume administered was 25µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d3). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
Positive control substance(s):
not specified
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogenous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) 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 differentes in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: In the mid and high dose group (10 and 50 %) the "positive level" which is 1.25 for the cell count index was just met (1.25) and exceeded (1.42). The stimulation indices for the weights of the draining lymph nodes were not affected..

Any other information on results incl. tables

Table 1: Summary of the LLNA/IMDS results (means of 6 animals per group)

Parameter investigated

Vehicle

control

Dose  1 %

 Dose  10 %

Dose  50 %

Stimulation index:

weight of draining lymph nodes

1.00

0.98

1.10

1.18

Stimulation index:

cell count in draining lymph nodes

1.00

1.16

1.25

1.42 *

Ear swelling in 0.01 mm on day 3 (index)

18.17

(1.00)

18.33

(1.01)

18.58

(1.02)

18.67

(1.03)

Ear weight in mg / 8 mm diameter punch on day 3 (index)

16.43

(1.00)

15.52

(0.94)

15.88

(0.97)

15.08

(0.92)

* increase exceeding "positive level" of 1.25

The mice did show a slight dose-dependent increase in the stimulation indices for cell counts but not for weight or ear swelling and ear weights. In the mid and high dose group (10 and 50 %) the "positive level" which is 1.25 for the cell count index was just met and exceeded, respectively. This classification is done despite the fact that a statistically significant increase is lacking. However, on the basis of lab experiences using this method, the "positive level" was set to an increase in cell count index by 0.25 (i.e. index >= 1.25). The "positive level" of ear swelling, which is 2x10-2 mm increase has never been reached in any case.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Executive summary:
Imidazotriazinon was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 1, 10 and 50 % formulated in polyethylene glycol 400 were tested. The results show that the test item has a weak sensitizing potential in mice after dermal application. Compared to vehicle treated animals there was an increase regarding the cell counts of the draining lymph nodes in the mid and high dose group. A significant difference compared to vehicle treated animals with respect to the weight of the draining lymph nodes as well as ear swelling was not reached in any case.

 

Thus, a hint for a substance specific activation of the cells of the immune system via dermal application was found by the method used, i.e. the test item has a weak skin sensitizing potential.