1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-09-25 to 2000-11-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

(1992)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

(1996)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A skin sensitization test according to OECD 406 has already excisted since 2000 and is sufficient for evaluation of the skin sensitisation potential of the test substance.

Test material

Test material information

Constituent 1

Reference substance name:

1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

EC Number:

259-393-4

EC Name:

1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

Cas Number:

54914-37-3

Molecular formula:

C18H34N2

IUPAC Name:

1,3,3-Trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethanamine

Details on test material:

Batch No. 00-0101, purity 97.2 %

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

- Strain: Dunkin Hartley (SPF quality)
- Source: Charles River Deutschland, Kisslegg (Germany)
- Age: approx. 5 weeks
- Weight at study initiation: 412 +/- 23 g
- Controls: 10 females (422 +/- 19 g); treatment: vehicle

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: see details below

Vehicle:

corn oil

Induction 2

Concentration / amount:

1st application: Induction 0.5 % intracutaneous
2nd application: Induction 20 % occlusive epicutaneous
3rd application: Challenge 10 % occlusive epicutaneous

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Challenge 2

Concentration / amount:

1st application: Induction 0.5 % intracutaneous
2nd application: Induction 20 % occlusive epicutaneous
3rd application: Challenge 10 % occlusive epicutaneous

No. of animals per dose:

20

Details on study design:

ADMINISTRATION/EXPOSURE 
- Preparation of test substance for induction: Within 4 hours prior to  treatment, homogeneous preparation with vehicle
- Induction schedule:
  Day 1: Injections
  Day 3: Assessment for dermal irritation
  Day 8: 48 hours occlusive patch with 0.5 ml of 20 % test substance on  clipped injection sites (control animals: vehicle)
  Day 10: Removal of patch and residual test substance, assessment for  dermal irritation
- Injection details: 0.1 ml each at 6 positions in clipped scapular  region:
  2 x Freund's Complete Adjuvant (FCA) / water for injection (50:50)
  2 x test substance 0.5 % in vehicle
  2 x test substance 1.0 % in vehicle / FCA (50:50)
  pairwise administration of each solution / suspension, symmetrical to  midline and from cranial to caudal
  controls: vehicle instead of test substance
- Challenge schedule:
  Day 22: Two 24 hours occlusive patches (each 0.15 ml) with
    (a) 10 % test substance and
    (b) vehicle alone on one clipped flank
  Day 23: Removal of patches and residual test substance
  Days 24 and 25: Assessment for challenge reaction 24 and 48 hours after  patch removal
- Rechallenge: no

Challenge controls:

10

Positive control substance(s):

yes

Remarks:

: alpha-hexylcinnamic aldehyde (tech. 85 %) (not more  than 6 months previously)

Results and discussion

In vivo (non-LLNA)

Results

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Eschar formation and/or scabs were seen in the majority of the treated skin sites among the experimental animals

Any other information on results incl. tables

RESULTS OF PILOT STUDY: 
  A) Intracutaneous: Erythema at > 0.1 %; necrosis at > 0.5 %
  B) Epicutaneous: At 24 hours reading erythema of grade 2 at 20 % and 50  %, necrosis and edema at 100 %. More serious findings in 50 % animals at  48 hours reading.
RESULTS OF TEST
- Sensitization reaction:
  Skin reactions of grades 1-3 in 20/20 animals both 24 and 48 hours  after removal of challenge patch. Additionally eschar formation and/or  scabs in most animals at 48 hours reading. No skin reactions were evident  in the control animals. 
- Clinical signs: No mortality occurred and no symptoms of systemic  toxicity were observed in the animals of the main study.
- Other: Mean body weight gain 145 g in test group, 129 g in control  group = same range

Applicant's summary and conclusion

Conclusions:

The skin reactions observed in response to a 10% test substance concentration in all experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals.
These results indicate a sensitisation rate of 100 per cent.

Executive summary:

The substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, twenty experimental animals were intradermally injected with a 0,5% concentration and epidermally exposed to a 20% concentration. Ten control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were challenged with a 10% test substance concentration and the vehicle. In the challenge phase, skin reactions varying between grades 1 and 3 were observed in all experimental animals in response to the 10% test substance concentration. No skin reactions were evident in the control animals. Eschar formation and/or scabs were seen in the majority of the treated skin sites among the experimental animals.

The skin reactions observed in response to a 10% test substance concentration in all experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 100 per cent. Therefore, the test substance may cause an allergic skin reaction.