Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-04-13 to 2006-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine)
EC Number:
619-422-7
Cas Number:
99305-42-7
Molecular formula:
C23 H42 N2 O4
IUPAC Name:
4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine)
Details on test material:
4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine) (H12MDU) of Degussa AG, batch 03.10.05/B-6320, purity 89.4 %

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: 
- Strain: CBA/Ca
- Sex: female
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD, Italy)
- Age: 8-12 weeks at time of treatment
- Weight at study initiation: 16.5-18.3 g
- Number of animals: 5 per dose group
- Controls: vehicle (negative)
- Housing: 5 to a cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 15 %
- Air changes (per hr): 15 to 20 per hour
- Photoperiod (hrs dark / hrs light): 12 h light/ 12 h dark


Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25, 50 % v/v
No. of animals per dose:
5
Details on study design:
1st application: Induction open epicutaneous
2nd application: Induction open epicutaneous
3rd application: Induction open epicutaneous
ADMINISTRATION/EXPOSURE 
- Preparation of test substance for induction: Suspension in vehicle,  concentrations 10%, 25%, and 50% v/v.
- Induction schedule:    
Days 1, 2, and 3: One daily topical application of 25 µl to the dorsum  of each ear.   
Day 6: Intraveneous treatment with 3H-methyl thymidine (3HTdR, 250 µl  of solution in sterile phosphate buffered saline = 20 µCi), 
5 hours later  sacrifice and processing of auricular lymph nodes.   
Day 7: Determination of 3HTdR incorporation.
- Concentrations used for induction: Each one group with 10%, 25%, and  50% (v/v)
- Positive control: alpha-hexylcinnamaldehyde (25% v/v in vehicle)
EXAMINATIONS   
Prior to sacrifice: Body weights (Days 1 and 6), 
clinical signs (once  daily)  
 ß-scintillation counting of auricular lymph node cell suspensions for  3HTdR incorporation as disintegrations per minute (DPM)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
STATISTICAL METHODS:   
Significance of differences amongst groups was assessed by analysis of variance. Differences between the treated group and the control group were assessed by Dunnetts test using a pooled error variance. Verification of homogeneity by Bartlett's test before Dunnetts test.   In case of inhomogeneity: Modified test (Cochran and Cox)  
Analysis of variance for differences amongst groups / Dunnett's test  for differences in comparison to the control group  
Stimulation index (SI) = ratio treated/control of disintegrations per  minute (DPM)   
Criteria: Sensitizing if SI >= 3 in any treatment group

Results and discussion

Positive control results:
In the group of treatment with hexyl cinnamic aldehyd the increase of DPM (9112.2 DPM) was clearly observed. The calculated SI was greater than
3 at the tested dose level (SI= 30.4).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The stimulation index (SI) was calculated as the ratio of the DPM/treatment group against the DPM/vehicle control group. SI: 16.36 (10% test item); 14.37 (25% test item); 5.09 (50%test item); 30.41 (pos. control)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Group               Mean DPM/animal       SI Vehicle                  299.6    10% test subst.        4902.8           16.36*    25% test subst.        4305.6           14.37*    50% test subst.        1525.0             5.09*    pos. control            9112.2           30.41*

Any other information on results incl. tables

RS-Freetext:
RESULTS OF TEST
- Sensitization reaction: 
  -----------------------------------------------
  Group               Mean DPM/animal       SI
  -----------------------------------------------
  Vehicle                 299.6
  10% test subst.        4902.8           16.36*
  25% test subst.        4305.6           14.37*
  50% test subst.        1525.0            5.09*
  pos. control           9112.2           30.41*
  -----------------------------------------------
  DPM = disintegrations per minute
  SI (stimulation index) = DPM (test) / DPM (vehicle)
  * p<0.05
  -----------------------------------------------
  The inverse concentration / effect relationship is tentatively assigned  to higher viscosity at higher concentrations leading to 

lower penetration.
- Clinical signs: No local irritation at application sites, no signs of  systemic effects.
- Body weights: Body weight development was within the expected range.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
According to the results of the study the test item H12MDU is considered to have the potential to cause skin sensitisation (delayed contact hypersensitivity) under the reported experimental conditions.
Executive summary:

The potential of the test item H12MDU, to induce and elicit delayed dermal sensitisation was assessed in the mouse using the LLNA test.

Five groups, each of 5 female mice, were treated with the test item at concentrations of 10%, 25%, 50%, with the reference item (alpha-hexylcinnamaldehyde) at the concentration of 25%, and with the vehicle alone.

Increases in 3HTdR incorporation were observed in animals treated with the test item when compared to the negative control group.

No signs of toxicity were observed following dosing animals at all concentrations, indicating a potential systemic effect of the test item. No local irritation was observed at the treatment sites.

Increases in 3HTdR incorporation were clearly recorded in comparison with the negative control group. The calculated SI was higher than 3 at all dose-levels tested. Reliability check was positive as expected.

On the basis of these results, the test item induced a greater than three fold increase in SI at all dose-levels tested. Therefore, it must be concluded that the test item H12MDU is considered to have the potential to cause skin sensitisation (delayed contact hypersensitivity) under the reported experimental conditions.