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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
Test has been performed before REACH regulation came into force requesting in vitro studies (October, 2016)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Apr 2012 to 13 Apr 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Up to 50% was tested because the substance is a solid at room temperature

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
other: Modified Local Lymph Node Assay: Integrated Model for the Differentiation of Skin reactions (IMDS)

Test material

Reference
Name:
Unnamed

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Remarks:
HsdWin:NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland, Kreuzelweg 53, 5960 AD Horst, Netherland
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 26 - 34 grams
- Housing: During the adaptation period up to 8 mice were housed together in conventional Makrolon ® type III cages. While during the study period the animals were single-housed in type II cages. Low-dust wood shavings named Lignocel BK 8-15 supplied by Rettenmaier & S6hne, GmbH & Co, 73494 Rosenberg, Germany were used as bedding.
- Diet: PROVIMI KLIBA SA 3883 maintenance diet for rats and mice (from Provimi Kliba SA, CH-4303 Kaiseraugst), ab libitum
- Water: tap water ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 -70
- Air changes (per hr): 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 and 50%
No. of animals per dose:
6
Details on study design:
JUSTIFICATION FOR SELECTED CONCENTRATIONS
Based on experience with the test system, the known properties of the test item and the solubility properties. The 50% was considered the maximum achievable concentration because the substance is a solid (when weighing the substance was heated and became a liquid).

METHOD
- 25 µL of the formulation was applied onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (D1, 2 and 3).
- The modifications in the LLNA method refer to the measurement of cell proliferation by cell counting instead of radioactive labeling.
- The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (day 4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
- The weights of the lymph nodes were determined on a Mettler semiautomatic balance
- After crushing the lymph nodes through a sieve in a 12-well plate, the cell counts per ml were determined using a Multisizer 3® from Coulter Electronics.

ANIMAL ASSIGNMENT AND TREATMENT
Criteria used to consider a positive response:
- A positive level for the cell count index is 1.4
- A positive level for the ear swelling test is 2 x E-2 mm increase (i.e. 10% of the control values).
- It has to be clarified that the "positive levels" mentioned above are exclusively defined for the NMRl outbreed mice used for this study

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item and positive control were formulated immediately before each administration in Acetone/Olive oil.

CLINICAL EXAMINATIONS
- The acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes
- Before the first treatment and before sacrifice the thickness of both auricles of the animals was measured using a spring-loaded micrometer (Oditest, Dyer Company or Fa. Kroeplin).
- On day 4 of the study the ear weight of the sacrificed animals was measured using a punch to take of a piece of every ear with a diameter of 8 mm. The weights were determined on a Mettler semiautomatic balance.
- The body weights of the animals were recorded at the start and the end of the study (day 1 and day 4):
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(s; vehicle) 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 nonparametric 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 differences in the means were calculated by Scheffe's method.

Results and discussion

Positive control results:
- After treatment with Alpha Hexyl Cinnamic Aldehyde (group 5) the NMRl mice showed increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts. compared to control animals, which are of no statistical significance. The "positive level", which is 1.4 for cell count indices, has been exceeded.
- The "positive level" of ear swelling has been exceeded in the positive control group.This increase is of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was detected, too.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Variability:
± 31.61%
Remarks on result:
other: 0% (vehicle control)
Key result
Parameter:
SI
Value:
1.03
Variability:
± 37.77%
Remarks on result:
other: 2%
Key result
Parameter:
SI
Value:
1.12
Variability:
± 33.96%
Remarks on result:
other: 10%
Key result
Parameter:
SI
Value:
1.24
Variability:
± 38.05%
Remarks on result:
other: 50%
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS:
- The positive level of ear swelling, which is 2E-2 mrn increase, i.e. about 10 % of the control values, has not been reached or exceeded in any dose group

BODY WEIGHTS
- The body weights of the animals were not affected by any treatment

Applicant's summary and conclusion

Interpretation of results:
other: Not sensitizing
Remarks:
in accordance with CLP (1272/2008 and its updates)
Conclusions:
Under the experimental conditions of this LLNA (OECD TG 429) study, the test item has no sensitising potential in mice after dermal application.
Executive summary:

The skin sensitisation potential of the test substance has been tested using a modified Local Lymph Node Assay (Integrated Model for the Differentiation of Skin reactions (IMDS)) according to OECD TG 429 and GLP principles. The modifications in the LLNA method refer to the measurement of cell proliferation by cell counting instead of radioactive labeling. The 50% is the maximum attainable concentration because the substance is a solid. The application of the test substance at concentrations of 2, 10 and 50% in a vehicle of acetone and olive oil (4:1 v/v) for three consecutive days did not result in an increased cell count above the (test system specific) positive level. In addition the positive level of ear swelling has not been exceeded in any dose group indicating the absence of irritating potential. No effects on body weight were observed. Based on the results, the substance was not considered to be a skin sensitiser.