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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 14, 2005 - April 8, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with EPA OPPTS 870.2600 and OECD Guideline 406 and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(butan-2-yl)-4-({4-[(butan-2-yl)amino]cyclohexyl}methyl)cyclohexan-1-amine
EC Number:
679-514-8
Cas Number:
154279-60-4
Molecular formula:
C21H42N2
IUPAC Name:
N-(butan-2-yl)-4-({4-[(butan-2-yl)amino]cyclohexyl}methyl)cyclohexan-1-amine
Details on test material:
- Name of test material (as cited in study report): Clearlink 1000
- Physical state: Clear liquid.
- Batch No.: CL-29720041.
- Expiration date of the lot/batch: July 2004.
- Specific activity (if radiolabelling): [methyl, 11,21-3H] Thymidine .
- Expiration date of radiochemical substance: March 2005.
- Storage condition of test material: Room temperature.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- CBA/J mice were supplied by Harlan Sprague-Dawley, Indianapolis
- Age at study initiation: 8 weeks
- Weight at study initiation: 17.8 - 23.6 g
- Housing: In polycarbonate with stainless steel wire cover cages individaully.
- Diet: PMI Feeds Inc. Formulab #5008 ad libitum.
- Water: Tap water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-12 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5, 5 or 10 % dilutions of test substance in acetone: olive oil vehicle.
No. of animals per dose:
5 females per dose group and 5 females in each control group.
Details on study design:
RANGE FINDING TEST:
Female mice were tested with three consecutive concentrations of test substance: 100% undiluted, and 50 and 25% v/v dilutions in acetone: olive oil. Clinical signs (activity decrease and swollen face) of toxicity were observed on Day 2, and two animals were found dead on Day 3. All remaining animals were sacrificed.

MAIN STUDY:
On days 1,2 and 3, each test animal in its group received an open application of 25 µl of an appropriate dilution (2.5, 5 or 10%) of the test substance to the dorsum of both ears. The vehicle control group (5 females) was treated in the same way as test animals, but with vehicle alone (acetone: olive oil) instead of test substance. The positive control group (5 females) was treated with 90% alpha-hexylcinnamaldehyde in acetone: olive oil. All test and control animals were given a two day rest period on Days 4 and 5.

On Day 6 of the study, test and control animals were injected in the tail vein with 250 µl of 0.01 M phosphate-buffered saline (PBS; Sigma, Lot 033K8201, Exp May 08), pH 7.4, containing 20 µCi of [methyl, 11,21-3H] Thymidine (Amersham Pharmacia, Lot 96, Exp Mar 05). Five hours after the injection, the animals were sacrificed, the draining auricular lymph nodes were excised and pairs from each individual animal were processed.

A single cell suspension was prepared by gently mechanical disintegration through 200 mesh stainless steel gauze. The cells were washed twice an excess of PBS and precipitated with 5% trichloroacetic acid (TCA; LabChem Inc, Lot 4142-26, Exp May 05) at 4°C for 18 hours. The pellets were resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid. incorporation of tritiated thymidine was measured by liquid scintillation counting as disintegrations per minute (DPM) from the paired lymph nodes of each animal, and mean DPM/animal was calculated for each group.

Individual body weights were recorded on Day 1 prior to dosing, and Day 6, prior to injection. All test and control animals were observed daily for clinical signs of toxicity and any signs of excessive irritation at the test site.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not required.

Results and discussion

Positive control results:
Positive control substance: alpha-hexylcinnamaldehyde in (80%) acetone: (20%) olive oil.
The positive control test substance produced a stimulation index of ≥3, and is therefore considered as a sensitiser.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
< 3
Test group / Remarks:
negative control
Parameter:
SI
Value:
7.5
Test group / Remarks:
2.5% test item concentration
Parameter:
SI
Value:
6.3
Test group / Remarks:
5% test item concentration
Parameter:
SI
Value:
7.2
Test group / Remarks:
10% test item concentration

Any other information on results incl. tables

Table 1. Group mean DPM.

Animal Group

Test Substance Concentration

Average Count per Mouse

Num. of Mice in Group

Test/Vehicle Control Ratio

Vehicle Control

NA

1132

5

NA

Test Group I

2,5%

8479

4

7,5

Test Group II

5%

7134

5

6,3

Test Group III

10%

8128

5

7,2

Positive Control

NA

3519

5

3,1*

NA= Not applicable

*= Positive Control used to confirm animal sensitization and validate procedures.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The potential sensitisation properties of Clearlink 1000 were tested with LLNA in mice. The test substance was administrated in aceton:olive oil with dose levels of 2.5, 5 and 10% to female rats (5/dose level). Vehicle control group and positive control group (5/animals per each control group) was included in the study. 1/5 rat in the medium dose level (5%) and 1/5 rat in the highest dose level (10%) lost weight during the study. Also in vehicle control group 1/5 and in positive control group 1/5 lost weight. One animal in the lowest dose group was found dead on day 6; otherwise all animals appeared normal for the duration of the study. The test substance produced a stimulation index of ≥3 in all groups of test animals. Based on the study results and it is considered as skin sensitizer.

The results of this study would lead to the classification of skin sensitisation (R43) in accordance with the criteria set in Directive 67/548/EEC. According to EU Regulation No. 1272/2008 (CLP), the classification would be skin sens. 1; May cause an allergic skin reaction.

The study is classified as acceptable and satisfies the guideline requirements for the skin sensitisation study. The result of this study is used as a key value in hazard assessment.