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Skin sensitisation

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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
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- modified LLNA = Local Lymph Node Assay (LLNA) - Integrated Model for Differentiation of Skin reactions (IMDS))
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Cell counts were measured instead of radioactive labelling. This provides similar sensitivity but the cell suspension can be further analysed with other methods, such as flow cytometry etc to gain insight in mechanistic events. Also, ear swelling was assessed. By comparing the specific immune reaction induced by the test item in the draining lymph nodes (LN cell counts/LN weights), with the immediate unspecific acute immune reaction (ear swelling/ear weight) it is possible to discriminate the irritant potential from the sensitizing potential of the compound tested. Modifications are authorized 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. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-Iminodiethanol, propoxylated
EC Number:
500-085-9
EC Name:
2,2'-Iminodiethanol, propoxylated
Cas Number:
35176-06-8
Molecular formula:
C4H11O2N(C3H6O)n; n = 1 – 6.5
IUPAC Name:
2,2'-Iminodiethanol, propoxylated
Details on test material:
- Physical state: liquid
- Content: > 99 %
- Stability under test conditions: analytically approved

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female

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:
Modified LLNA
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation and 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 lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling
- ear weight
- 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 (3%, 10% and 30%), checked in regular intervals, shows a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Compared to vehicle-treated animals there were clear increases regarding the draining lymph nodes and cell counts in the high dose group. the increases are of statistical significance. The cell count indices were determined to be 01.03 (2 %), 1.01 (10 %) and 1.84 (50 %). Thus the "positive level", which is 1.4 for the cell count index, was clearly exceeded at the high dose group. Differentiation indices (DI) which is the quotient of the relative lymph node reaction divided by the relative acute skin reaction were > 1 for the high dose group, i.e. 3.5 (for further information see: Homey et al., Toxicol. and Appl Pharmacol. 153, 83-94 (1998) and Vohr et al., Arch. Toxicol., 73, 501-509 (2000)). This DI value does also point to a skin sensitizing potential.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labeling
Parameter:
other: cell count indices
Value:
1.03
Test group / Remarks:
2%
Parameter:
other: cell count indices
Value:
1.01
Test group / Remarks:
10%
Parameter:
other: cell count indices
Value:
1.84
Test group / Remarks:
50%
Remarks on result:
other: Thus the "positive level", which is 1.4 for the cell count index, was clearly exceeded at the high dose group
Parameter:
other: Differentiation indices (DI)
Remarks:
quotient of the relative lymph node reaction divided by the relative acute skin reaction
Value:
3.5
Test group / Remarks:
50%
Remarks on result:
other: DI > 1. This DI value does also point to a skin sensitizing potential.

Any other information on results incl. tables

The "positive level" of ear swelling which is 2 x 10-2 mm increase, i.e. about 10% of the control values, has not been reached or exceeded in any dose group. Nevertheless a statistically significant increase has been detected after administration of the high concentration of the test item in ear weights. Although this effect is not corroborated by increase in ear swelling an irritant potential can not be excluded.

It has to be clarified that the "positive levels" mentioned above are exclusively defined for the NMRI outbred mice used for this study. Such positive limits have to be calculated for each strain of mice individually.

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

Applicant's summary and conclusion

Interpretation of results:
other: The EC 1.4 value calculated is 28.80%. In accordance with the classification proposed in the ECETOC TR No. 78 this value corresponds to a weak skin sensitizer.
Conclusions:
The EC 1.4 value calculated is 28.80%. In accordance with the classification proposed in the ECETOC TR No. 78 this value corresponds to a weak skin sensitizer.
Executive summary:

Diethanolamine, propoxylated showed a weak sensitizing potential in the modified Local Lymph Node Assay (IMDS) in female NMRI mice after dermal application of a 50 % concentration. Additionally, an irritant potential cannot be excluded because an increase in ear weights but no increase regarding ear swelling was described in the high dose group. Based on these results, classification of Diethanolamine, propoxylated as skin sensitiser is not warranted.

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