Registration Dossier

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: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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 (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Desmodur VP.PU 60WF14
- Physical state: liquid
- Purity: 100 % (as indicated by the sponsor)
- Lot/batch No.: P1DE000345
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland, Horst, Netherlands
- Strain: Hsd Win:NMRI
- Age at study initiation: 9 weeks
- Weight at study initiation: 25 - 34 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 2, 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 (d1, d2 and d3). 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 (d4). 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)
- body weights
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 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

Positive control results:
Alpha hexyl cinnamic aldehyde, checked in regular intervals, showed a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The results show  that the test item has a sensitizing potential in mice after dermal application. Compared to vehicle treated animals there was a clear increase in weights of the draining lymph nodes (indices of 4.24, 4.74 and 3.74 %, resp.) and in the cell counts (indices of 4.29, 4.66 and 3.67 %, resp.) at dose groups of 2, 10 and 50 %. These changes are of statistical significance. The "positive level", which is 1.4 for cell counts, has been exceeded in all dose groups.

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  2 %

 Dose  10 %

Dose  50 %

Stimulation index:

weight of draining lymph nodes

1.00

4.24 *

4.74 *

3.74 *

Stimulation index:

cell count in draining lymph nodes

1.00

4.29 *

4.66 *

3.67 *

Ear swelling in 0.01 mm on day 4 (index)

17.50

(1.00)

 22.42 *

(1.28)

 29.25 *

(1.67)

 36.67 *

(2.10)

Ear weight in mg / 8 mm diameter punch on day 4 (index)

 11.57

(1.00)

 14.75 *

(1.28)

 19.51 *

(1.69)

 24.46 *

(2.11)

* statistically significant increase (p ≤ 0.05)

The "positive level" of ear swelling, which is 2 x l0-2 mm increase, has been exceeded in all dose groups. An increase in this parameter would point to an acute irritant (inflammatory) response. However, such an irritant property could also be combined with a strong skin sensitizing potential of a test compound. Nevertheless, it must be concluded that the test item has an irritant potential at all concentrations tested. Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 1%. In accordance with the classification proposed in the Technical Report No.78 of ECETOC this value corresponds to at least a strong skin sensitizer.

The body weights of the animals were not affected by any treatment.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Executive summary:

Desmodur VP.PU 60WF14 was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 2, 10 and 50% formulated in acetone/olive oil (4:1) were tested. The results show that Desmodur VP.PU 60WF14 has a strong sensitizing potential in mice after dermal application. Compared to vehicle treated animals there was a significant increase regarding the weights of the draining lymph nodes and the cell counts in all dose groups. The corresponding cell count indices were 4.29, 4.66 and 3.67 exceeding the "positive level" of index 1.4. A significant increase compared to vehicle treated animals regarding ear swelling and ear weights was detected in all dose groups. An increase in this parameter would point to an acute irritant (inflammatory) response. However, such an irritant property can also be combined with a strong skin sensitizing potential of a test compound.