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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
Study period:
2005-07-26 to 2005-11-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Principles of method if other than guideline:
Cell count was performed using electric field multi-channel cell counting system (Casy-Counter), non-radioactive variant.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile
EC Number:
300-496-1
EC Name:
3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile
Cas Number:
93940-97-7
Molecular formula:
C16H28N4
IUPAC Name:
3-[(3-{[(2-cyanoethyl)amino]methyl}-3,5,5-trimethylcyclohexyl)amino]propanenitrile
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: V3186/11

Letter of Confirmation: CAS No 90530-15-7 and 93940-97-7 refer to the same substance and substance composition.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann GmbH, Gartenstr. 27, 33178 Borchen, Germany
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 16.3 g - 19.5 g
- Housing: The single housed; Makrolon type I cages
- Diet: Kliba Labordiät (Maus / Ratte Haltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: 15 days before the first test substance application.
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): fully air conditioned room
- Photoperiod (hrs dark / hrs light): light (6.00 a.m. - 6.00 p.m.) 12 h darkness (6.00 p.m. - 6.00 a.m.)

Study design: in vivo (LLNA)

Vehicle:
other: acetone
Concentration:
0.3%, 1 %, 10% (w/w)
No. of animals per dose:
6
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Acetone was used as the vehicle because good solubility of the preparation was achieved.
- Systemic toxicity: No signs of systemic toxicity were noticed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
If a test substance does not show a statistically significant and/or biologically relevant increase in cell count and/or lymph node weight as compared to the vehicle control in the presence of statistically significantly and/or biologically relevant increased ear weights as indication of skin irritation, it is considered not to be a sensitizer.
If at least one concentration tested causes a concentration dependent statistically significant and/or biologically relevant increase in cell count and/or lymph node weight without being accompanied by a statistically significant and/or biologically relevant increase in ear weight, the test substance is considered to be a sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
Application volume: 25 µL per ear
Site of application: Dorsal part of both ears
Frequency of application: 3 consecutive applications (day 0 - day 2) to the same application site
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The indices of lymph node weight, cell count and ear weight were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
Statistical Test for the parameters lymphnode weight, cell count, and ear weight: WILCOXON -Test.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control
Key result
Parameter:
SI
Value:
1.19
Test group / Remarks:
0.3 %
Remarks on result:
other: based on cell count
Key result
Parameter:
SI
Value:
1.08
Test group / Remarks:
1 %
Remarks on result:
other: based on cell count
Key result
Parameter:
SI
Value:
3.6
Test group / Remarks:
10 %
Remarks on result:
other: based on cell count
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Treatment of the mice with a 10% test substance preparation induced statistically significant increase in lymph node cell counts as compared to the vehicle control group. The lymph node weights were statistically significant increased and therefore in congruency with the cell counts.

DETAILS ON STIMULATION INDEX CALCULATION
test group x / test group 1 (vehicle control )

EC3 CALCULATION
not applicable

CLINICAL OBSERVATIONS:
No abnormalities were observed during general observation.

BODY WEIGHTS
The expected body weight gain was generally observed in the course of the study.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria