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Description of key information

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:
February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2010). 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).
Deviations:
yes
Remarks:
- Measurement of cell counts instead of radioactive labeling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Stability under test conditions: The stability of the substance in the formulation was analytically verified for at least 4 days.
- The substance in the test item is dissolved in approx. 30 % solvent. Test concentrations in the skin sensitization study were adjusted to the substance content (approx. 70 %).
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdWin: NMRI (SPF-bred)
- Source: Harlan Nederland, 5960 AD Horst, The Netherlands
- Age at study initiation: 8 weeks
- Weight at study initiation: 26-36 g
- Housing: During the study period the animals were single-housed in type II cages.
- Diet and 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
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 (vehicle control), 2, 10, 50 % (due to its viscosity it was not possible to pipette the test item pure)
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation was 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 25 µl/ear. The concentrations used were based on the experiences with the test system and the properties of the test substance. For negative control a dose group treated only with the vehicle in the above described manner was used.
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).
The following endpoints were determined:
- weight of draining lymph nodes (relative to vehicle control)
- cell counts in draining lymph nodes (absolute and relative to vehicle control)
- ear swelling (Day 1 absolute, Day 4 absolute and relative to vehicle control)
- ear weight (based on a 8 mm diameter piece punched from the ears; Day 4 absolute and relative to vehicle control)
- body weights (Day 1 and 4, absolute)

The relative indices are calculated by dividing the values of the test groups by the values of the vehicle control group, e.g. cell counts of substance-treated lymph nodes divided by cell counts of vehicle-treated lymph nodes. Thus, in case of no stimulating effect the index is about 1.00 (± relative standard deviation). By comparing the specific immune reaction induced by the test item in the draining lymph nodes (LN cell counts / LN weights) with the immediate non-specific acute skin reaction (ear swelling / ear weight) this IMDS method allows for discrimination of the irritant potential from the sensitizing potential of the compound tested.
The “positive level” of 1.4 for relative cell counts is exclusively defined for the NMRI outbreed mice used in this study. Such positive limits have to be calculated for each strain of mice individually.

The methodological reliability is checked in regular intervals using alpha cinnamic aldehyde as positive control. The closest reliability test using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentrations of 2.5 %, 10 % and 40 % clearly showed the sensitizing potential of the test item.
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 differences in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.
Positive control results:
After treatment with Alpha Hexyl Cinnamic Aldehyde mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to control animals. These increases were of statistical significance.
Parameter:
SI
Remarks:
relative Cell Count (compared to vehicle control)
Value:
1.04
Test group / Remarks:
50 % (content adjusted)
Remarks on result:
other: The cut-off for cell count of 1.4 was never reached or exceeded in any dose group: 1.00 (0 %) / 0.93 (2 %) / 1.10 (10 %) / 1.04 (50 %)
Cellular proliferation data / Observations:
BODY WEIGHTS
The body weights of the animals were not affected by the treatment.

The mice did not show increases for weights or cell counts of the draining lymph nodes after application of the test item:

Cell count indices: 1.00 (0 %) / 0.93 (2 %) / 1.10 (10 %) / 1.04 (50 %), Weight index: 1.0 (0%), 0.93 (2%), 1.06 (10%), 1.06 (50%).

The positive level of ear swelling, which is 2 x 10exp-2, i.e. about 10 % of the control values, has been exceeded in the high dose group (ear swelling [mm x 10 exp-2]: day 1 = 18.25 (0 %) / 17.92 (2 %) / 18.17 (10 %) / 17.92 (50 %); day 4 = 19.00 (0 %) / 19.58 (2 %) / 20.25 (10 %) / 32.83 (50 %); Index day 4 = 1.00 (0 %) / 1.03 (2 %) / 1.07 (10 %) / 1.73 (50 %)).

The ear weights are statisticallly significant increased in the high dose group (day 4 = 14.05 (0 %) / 13.35 (2 %) / 14.68 (10 %) / 20.89 (50 %); Index day 4 = 1.00 (0 %) / 0.95 (2 %) / 1.04 (10 %) / 1.49 (50 %)).

Although an increase in ear swelling might point to an irritating potential the increase in ear weight is most properly due to substance residues still remaining on the ear. The lack of ear skin reddening as well as lack of increase in LN weights also supports this interpretation.

Interpretation of results:
other: no skin sensitisation potential
Executive summary:

A modified IMDS-LLNA (Integrated Model for the Differentiation of Skin Reactions; OECD TG 429) was performed on 6 female NMRI mice per dose group using substance formulations of 0 % (vehicle control), 2 %, 10 % and 50 % (due to its viscosity it was not possible to pipette the test item pure; the content of the substance in the test item, which is approx. 70 % in 30 % solvent, was considered). The IMDS method allows for discrimination of the irritant potential by comparing the specific immune reaction induced by the test substance in the draining lymph nodes (LN cell counts / LN weights) with the immediate non-specific acute skin reaction (ear swelling / ear weight).

Compared to vehicle (A/OO) treated animals there was no increase regarding the weights or the cell counts of the draining lymph nodes. The "positive level" of the stimulation index for cell counts, which is 1.4, has not been exceeded in any dose group. The "positive level" of ear swelling, which is an increase of 2 x 10exp-2 mm, i.e. about 10 % of the control values, has been exceeded in the high dose group. In addition, the ear weights of the high dose group were significantly higher compared to control animals. Although an increase in ear swelling might point to an irritant potential the increase in ear weight is most probably due to substance residues remaining on the ear. A lack of ear skin reddening as well as a lack of increase in LN weights supports this interpretation.

In conclusion, the study results did not reveal indications for a skin sensitizing potential up to and including a 50 % concentration of the substance, and also no conclusive indications for an irritant potential were found. 50 % turned out to be the NOEL in this study with respect to skin sensitization.

 

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

For the assessment of skin sensitisation a modified IMDS-LLNA (Integrated Model for the Differentiation of Skin Reactions) according to OECD TG 429 is available. In this study 6 female NMRI mice per group were treated with substance formulations of 0 % (vehicle control), 2 %, 10 % and 50 % (due to its viscosity it was not possible to pipette the test item pure; the content of the substance in the test item, which is approx. 70 % in approx. 30 % solvent, was considered). The IMDS method allows for discrimination of the irritant potential from the sensitizing potential by comparing the specific immune reaction induced by the test substance in the draining lymph nodes (LN cell counts / LN weights) with the immediate non-specific acute skin reaction (ear swelling / ear weight).

Compared to vehicle (A/OO) treated animals there was no increase regarding the weights or the cell counts of the draining lymph nodes. The "positive level" of the stimulation index for cell counts, which is 1.4, has not been exceeded in any dose group. The "positive level" of ear swelling, which is an increase of 2 x 10exp-2 mm, i.e. about 10 % of the control values, has been exceeded in the high dose group. In addition, the ear weights of the high dose group were significantly higher compared to control animals. Although an increase in ear swelling might point to an irritant potential the increase in ear weight is most probably due to substance residues remaining on the ear. A lack of ear skin reddening as well as a lack of increase in LN weights supports this interpretation.

In conclusion, the study results did not reveal indications for a skin sensitizing potential up to and including a 50 % concentration of the substance, and also no conclusive indications for an irritant potential were found. 50 % turned out to be the NOEL in this study with respect to skin sensitization.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for skin sensitisation up to a 50 % concentration.