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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
19 Nov - 22 Nov 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions. Modified Local Lymph Node Assay (Integrated Model for the Differentiation of Skin Reactions, IMDS) accordimng to Vohr, H.-W. et al. (Arch Toxicol 73:501-509, 2000) and Ehling, G. et al. (Toxicology 212: 60-68 and 89-79, 2005).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Modified Local Lymph Node Assay (Integrated Model for the Differentiation of Skin Reactions, IMDS) accordimng to Vohr, H.-W. et al. (Arch Toxicol 73:501-509, 2000) and Ehling, G. et al. (Toxicology 212: 60-68 and 89-79, 2005).
GLP compliance:
yes
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): only trade name given
- Chemical name: Acetylided glyceride-mixture
- Physical state: yellowish, clear liquid
- Analytical purity: 99.8%
- Lot/batch No.: CH70920C
- Expiration date of the lot/batch: 20 Sep 2008
- Stability under test conditions: the stability of the test item in the vehicle was analytically verified for up to 4 days. The test item was shown to be stable in the vehicle for at least this period of time.
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: Hsd Win:NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 9 weeks
- Weight at study initiation: 23-29 g
- 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. During adaptation period, the cages were changed at least twice a week. Low-dust wood shavings were used as bedding.
- Diet: PROVIMI KLIBA SA 3883 maintenance diet for rats and mice (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

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

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2, 10 and 50%
No. of animals per dose:
6
Details on study design:
RANGE FINDING TESTS:
A range finding test was not conducted. According to the author, based on in-house experience with the test system and the available information on the properties of the test item, the following concentrations were used: 0 (vehicle control), 2, 10 and 50%."

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node weight and cell count determinations
- Criteria used to consider a positive response:
Cell count index: The "positive level" for the cell count index was stated to be 1.4.
Ear swelling: The "positive level" of ear swelling was stated to be a 2E-02 mm increase (about 10% of the control values)
The author explicitly stated that the cut-off values for "positive levels" mentioned above are exclusively defined for the NMRI outbred mice used for this study (Homey, B. et al. [1998] Toxicol. and Appl. Pharmacol. 153:83-94; Vohr, H.-W. et al. [2000] Arch. Toxicol. 73:501-509). Further, the author noted that such positive limits have to be calculated for each strain of mice individually (Ehling, G. et al. [2005] Toxicology 212:60-68; Ehling, G. et al. [2005] Toxicology 212:69-79).

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation or the vehicle 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.

INVESTIGATIONS

-Autopsies:
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).
- LLN Weight and cell count determinations:
The weight of the lymph nodes was determined on a semiautomatic balance and stored electronically. After crushing the lymph nodes through a sieve in a 12-well plate and dispersion in 2 mL PBS, the cell counts per mL were determined using an electronic cell counter. These data were also directly collected and processed by a computer. Mean values, indices and standard deviations were calculated by a spreadsheet software.
A special software was used to calculate mean values and standard deviations of lymph node weights. Indices were calculated manually.
The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance-treated lymph nodes by the vehicle-treated ones. Thus, in case of no stimulating effect, the index is always about 1.00 (± standard deviation), and the indices of vehicle-treated animals are set to 1.00 (± standard deviation).
- Ear swelling:
Before the first treatment and before sacrifice, the thickness of both auricles of the animals was measured using a spring-loaded micrometer. Mean values, indices and standard deviations of the ear swelling were calculated by a spreadsheet software.
- Ear weight:
On Day 4 of the study, the ear weight of the sacrificed animals was measured using a punch to take a piece of every ear with a diameter of 8 mm. The weights were determined on a semiautomatic balance. Mean values, indices and standard deviations of the ear weights were calculated by a spreadsheet software.
- Body weight:
The body weights of the animals were recorded at the start and at 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 the treated groups were compared with those from the control group(s; vehicle) by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances were considered heterogeneous (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 Scheffe'S method, which can be used for both equal and unequal sample sizes.
In this method of statistical processing of measurements a large number of comparisons is made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems, the biological and toxicological relevance was also taken into consideration in the evaluation of statistical significance.
For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values were used in the evaluation of the biological relevance.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Cell count index (± standard deviation in %): Vehicle control: 1.00 ± 27.43 2%: 0.84 ± 32.82 10%: 0.95 ± 20.12 50%: 0.97 ± 20.22 Ear swelling index on Day 4: Vehicle control: 1.00 2%: 1.04 10%: 1.00 50%: 1.02 Ear weight index: Vehicle control: 1.00 2%: 0.99 10%: 1.00 50%: 1.06
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
No significant changes were observed in the absolute number of lymph node cell counts per mL (see Table 2 for details) Group mean cell counts (1000 cells/mL lymph node suspension ± standard deviation): Vehicle control: 10961,50 ± 3006.68 2%: 9169.42 ± 3009.43 10%: 10421.25 ± 2096.75 50%: 10629.50 ± 2149.53 No significant changes were observed in ear thickness between Day 1 and Day 4 as well as between animals treated with the test item and with the vehicle (see Table 3 for details). Group mean ear swelling (Day 1 / Day 4; in 0.01 mm ± standard deviation in %): Vehicle control: 17.83 ± 3.24 / 17.25 ± 2.62 2%: 17.67 ± 3.69 / 17.92 ± 2.87 10%: 17.08 ± 3.01 / 17.17 ± 4.86 50% 17.33 ± 5.12 / 17.67 ± 4.41

Any other information on results incl. tables

Table 2. Individual cell counts

Cell count (1000 cells/mL lymph node suspension)
Treatment group Animal No. Arithmetic mean (n = 2) Group mean SD SD (%) Cell count index
Vehicle 1 10541.5 10961.50 3006.68 27.43 1.00
2 13500.5
3 11383
4 11254.5
5 13686
6 5403.5
2% 7 7443 9169.42 3009.43 32.82 0.84
8 9853.5
9 9090.5
10 6253.5
11 14736
12 7640
10% 13 10796.5 10421.25 2096.75 20.12 0.95
14 9343
15 7629.5
16 14002
17 10206.5
18 10550
50% 19 7714.5 10629.50 2149.53 20.22 0.97
20 8873.5
21 12331.5
22 11729.5
23 13247.5
24 9880.5

Table 3. Ear swelling (6 animals per group, in 0.01 mm).

Dose (%) Day 1 (mean ± SD in %) Day 4 (mean ± SD in %) Index Day 4
0 (vehicle) 17.83 ± 3.24 17.25 ± 2.62 1.00
2 17.67 ± 3.69 17.92 ± 2.87 1.04
10 17.08 ± 3.01 17.17 ± 4.86 1.00
50 17.33 ± 5.12 17.67 ± 4.41 1.02

Table 4. Ear and lymph node weight.

Ear weight (6 animals per group, in mg per 8 mm punch) Lymph node weight (6 animals per group)
Dose (%) Day 4 (mean ± SD in %) Index Day 4 Weight index (index of mean ± SD in %)
0 (vehicle) 11.44 ± 6.52 1.00 1.00 ± 20.70
2 11.28 ± 4.64 0.99 0.99 ± 28.19
10 11.43 ± 9.14 1.00 1.00 ± 23.44
50 12.18 ± 7.94 1.06 1.02 ± 20.02

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified