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Toxicological information

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
24 April 2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: "Xyligen LP 15670".
Batch No.: VM 2008.
Appearance: Yellowish liquid.
Purity: 30.41%
Solubility: Miscible with water in any proportion
Density: About 1.1 kg/I.
pH: About 10.
Specific details on test material used for the study:
Name: "Xyligen LP 15670" (N-Cyclohexyldiazeniumdioxy-potassium (as aqueous solution)).
Batch No.: VM 2008.
Appearance: Yellowish liquid.
Purity: 30.41%
Solubility: Miscible with water in any proportion
Density: About 1.1 kg/I.
pH: About 10.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Test system (animals):
Species, strain: Mice, CBA/CaOlaHsd.
Supplier: Harlan Winkelman GmbH, D-33176 Borchen.
Sex, specification: Females, healthy young adult nulliparous animals.
Age of the animals: About 8 weeks at the first administration.
Weight range of the animals at the first application: 17.6 - 21.8 g.
Number of animals: 6 animals/group (including spare animals): 18 animals for 3 test substance groups, 6 animals for the negative control group, 6 animals for the positive control group.
Spare animals: The 6th animal of each group served as spare animal. It was treated in the same way as the other animals of its group. Since all animals survived and all animals received the correct amount of 3 HTdR all spare-animals were taken for the examination of the results. The number of animals/group was then 6.

Animal maintenance:
Hygiene: Optimal hygienic conditions.
Room No.: EH 1-15.
Room temperature: Average of 21.9°C (continuous monitoring and recording).
Relative humidity: Average of 52.3 % (continuous monitoring and recording).
Light: Only artificial light from 6.00 a.m. to 6.00 p.m.
Cages: Single caging. Makrolon cages type 11, (22 cm x 16,5 cm ground area, 15 cm high).
Feed: Altromin maintenance diet for rats and mice, item No. 1324forte, ad libitum. Random samples of the feed are analysed for contaminants by Altromin, D-32791 Lage.
Water: Tap water offered in Makrolon-bottles with stainless steel canules, ad libitum.
Bedding material: Wood chips (aspen) Fa. ABEDD Dominik Mayr KEG, A-8580 Koflach. Reduction of microorganisms by autoclaving.
Acclimatisation: 7 days.
Identification of the animals: By shearing a defined fur region on the back of the animal:
Group A: top left
Group B: top right
Group C: bottom left
Group P: top left and top right
Group K: unshorn and by cage tag.



Study design: in vivo (LLNA)

Vehicle:
other: water
Remarks:
Solubility testing of the test substance in the guideline recommended solvents showed that the test substance is not soluble in any of the guideline recommended solvents
Concentration:
50%, 25% and 10% of test substance
No. of animals per dose:
6 animals per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: the test substance is only soluble in water
- Irritation: not observed
- Systemic toxicity: slightly reduced motoric activities with 50 % test substance; no signs of systemic toxicity with 25 % and 10 % test substance observed
- Ear thickness measurements: No increase in ear thickness and no local irritations at the application sites could be observed in all animals treated with 50%, 25% and 10%.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The calculated stimulation indices (test substance/negative control ratio) were decisive for the grading of the potential of sensitisation: According to the guidelines the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response. The Sis of the tested test substance concentrations were 1.5 (low dose), 2.4 (mid dose) and 1.9 (high dose).

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was diluted in water and was administered to three groups of 6 female CBA/Ca mice. Administration was performed epicutaneously to the dorsal surface of both ears, once a day on three consecutive days. The volume administered was 25 μL per ear.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Analysis of variance followed by the Scheffé test: dpm data, comparisons between dosed groups and the negative control group
t-test: dpm data for comparison of two groups only
P = 0.05 was chosen in each test. Two tailed tests were used. Numerical data have been rounded for presentation, a manual recalculation therefore may yield slightly different results to those given in the tables.

Results and discussion

Positive control results:
Application of 25% HCA in acetone : olive oil (4:1, v/v) resulted in an Stimulation index of 53.3. This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
low dose group
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
mid dose group
Key result
Parameter:
SI
Value:
1.9
Test group / Remarks:
high dose group

Any other information on results incl. tables

General

All animals survived till the end of the study. No adverse effects were noted in all animals of the groups A, B, K and P and in 3 animals of group C throughout the whole study. 3 animals of the high dosed group C showed on days 3-5 reduced motoric activities. Hunshed positions were observed in one animal on Day 3 and in 2 animals on Day 4 .

Body masses and body mass gains of all animals were in the range to be expected from animals of the same strain, sex and age. No skin irritating effects were observed in all animals of the groups A, B, K and P and in 4 animals of group C throughout the whole study. 2 Animals of the high dosed group C had white crusts at the application sites on Days 3-5.

3H-thymidine incorporation, stimulation indices

The calculated stimulation indices (test substance/negative control ratio) were decisive for the grading of the potential of sensitisation: According to the guidelines the decision process with regard to a positive response includes a stimulation index of equal to or greater than 3, together with consideration of dose-response. The SIs of the tested test substance concentrations were 1.5 (low dose), 2.4 (mid dose) and 1.9 (high dose).

Positive control

The positive control substance led to a stimulation index of 53.3 thus demonstrating the validity of the experiment.

Applicant's summary and conclusion

Conclusions:
According to the OECD-Guideline 429, the Directive 2004/73/EC, method B.42., "Skin Sensitisation: Local Lymph Node Assay" and the EPA Guideline OPPTS 870.2600, "Skin Sensitization", March 2003 "Xyligen LP 15670" is regarded as a non-sensitiser in the LLNA.
Executive summary:

"Xyligen LP 15670" is regarded as a non-sensitiser in the LLNA according to the OECD Guideline 429, the Directive 2004/73/EC, method 8.42., "Skin Sensitisation: Local Lymph Node Assay" and the EPA Guideline OPPTS 870.2600, "Skin Sensitization" since the stimulations indices of all examined test substance concentrations were clearly smaller than 3.