Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin sens: OECD 429; NMRI mouse; not sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
See further explanation in "Principles of method if orther than guideline" below.
Principles of method if other than guideline:
The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.
In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method (Ehling et al. 2005a and Ehling et al. 2005b)).
Ehling et al. 2005a: Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
Ehling et al. 2005b: Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).

GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories,Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 9 weeks
- Weight at study initiation: 26-32 g
- Housing: kept individually in MAKROLON cages (type III)
- Diet (e.g. ad libitum): Commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany; served as food ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 12-18 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
(3+1 v/v used instead)
Concentration:
5 % active substance
10 % active substance
25 % active substance
No. of animals per dose:
6 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: soluble
- Irritation: not observed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method (Ehling et al. 2005a and Ehling et al. 2005b)

Ehling et al. 2005a: Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
Ehling et al. 2005b: Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).


TREATMENT PREPARATION AND ADMINISTRATION:
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible irritating potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers would have been determined according to the Nalimov test.
Positive control results:
The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.838
Test group / Remarks:
positive control
Remarks on result:
other: Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.139
Test group / Remarks:
25% concentration
Remarks on result:
other: Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.067
Test group / Remarks:
10% concentration
Remarks on result:
other: Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive
Parameter:
SI
Remarks:
Lymph node cell count
Value:
0.971
Test group / Remarks:
5% concentration
Remarks on result:
other: Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive
Key result
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1
Test group / Remarks:
negative control
Remarks on result:
other: Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive

 


Stimulation indices (SI)*:















































Parameter



Group 1, negative control



Group 2,


5%



Group 3, 10%



Group 4, 25%



Group 5,


positive control



Lymph node cell count



1.000



0.971



1.067



1.139



1.838



Lymph node weight



1.000



0.948



1.172



1.103



1.431



Ear weight



1.000



1.006



1.043



0.933



1.171



Difference of


ear thickness



1.000



1.009



0.966



0.974



1.265




____ significantly different from control at p ≤ 0.01


*Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method (Ehling et al. 2005a and Ehling et al. 2005b)

Interpretation of results:
GHS criteria not met
Remarks:
EU GHS criteria not met
Conclusions:
In conclusion, under the present test conditions, IPETC at concentrations of 5%, 10% or 25% (w/w) in acetone/olive oil (3+1, v/v) did not reveal any sensitising properties in the local lymph node assay.
Executive summary:

An in vivo skin sensitization LLNA test in female mice with the test substance at concentrations of 5%, 10% or 25% (w/w) in acetone/olive oil (3+1, v/v) was performed equivalent and similarly with OECD Guideline 429. In the current study, instead of a radioactive method an alternative method was used, employing the lymph node weight and lymph node cell count to assess proliferation. Stimulation indices were consequently calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones. Based on the obtained test results and in accordance with the criteria of Classification, Labelling and Packaging of Substances and Mixtures Regulation (EC) No 1272/2008 (CLP Regulation), the test substance is not classified as skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Treatment with IPETC at concentrations of 5%, 10% or 25% did not reveal statistical significantly increased values for lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising. No effect was observed in the lymph node weight, either.


The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted.


The positive control group caused the expected increases in lymph node cell countand lymph node weight(statistically significant atp ≤ 0.01).Therefore, the study can be regarded as valid.


No signs of local or systemic intolerance were recorded. The animal body weight of the high dose group is reduced compared to all other groups.


In conclusion, under the present test conditions, IPETC at concentrations of 5%, 10% or 25% (w/w)inacetone/olive oil (3+1, v/v)did not reveal any sensitising properties in the local lymph node assay.




Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

As for the substance no positive results for skin sensitisation are shown from an appropriate animal test, substance does not apply to classification according to the criteria of Classification, Labelling and Packaging of Substances and Mixtures Regulation (EC) No 1272/2008 (CLP Regulation), and is therefore not classified as such.