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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 1990 - 09 August 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
12 May 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
September 1984
Deviations:
no
Principles of method if other than guideline:
"Experimental Skin Sensitization in the Guinea Pig and Man", Buehler E. V. and Griffith F. in: Animals
models in dermatology (ed. H.I. Maibach), pp. 56-66, Edinburgh, Churchill Livingstone, 1975.
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
An appropriate Buehler test is available which would not justify conducting an additional LLNA due to animal welfare.

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea
EC Number:
406-370-3
EC Name:
3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea
Cas Number:
58890-25-8
Molecular formula:
C27 H36 N4 O2
IUPAC Name:
1-cyclohexyl-3-[4-({4-[(cyclohexylcarbamoyl)amino]phenyl}methyl)phenyl]urea
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): MDI/CHA
- Physical state: white yellowish solid
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Himalayan albino
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: 299 - 409g
- Housing: Group housing of 2 animals per cage with wire-mesh floors.
- Diet: Free acces to standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4mm (Hope farms, Woerden, The Netherlands). In addition, hay was provided once a week.
- Water: Free access to tap-water, diluted with decalcified water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 02 July 1990 To: 09 August 1990

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
25% (w/w) / 0.5 mL
Day(s)/duration:
6 hours on days 1, 3, 5, 8, 10, 12, 15, 17 and 19
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
25% (w/w) / 0.5 mL
Day(s)/duration:
6 hours on day 29
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
25%, 10% and 5% (w/w) / 0.05 mL
Day(s)/duration:
6 hours on day 36 (1 week after the first challenge)
Adequacy of challenge:
other: series of three concentrations
No. of animals per dose:
Treatment group: 20
Control group: 10
Details on study design:
PRIMARY IRRITATION EXPERIMENT:
A primary irritation study was conducted in order to select test item concentrations suitable for the induction and challenge phase in the main study.
One animal was treated with 0.5 mL test item with a concentration of 50% (w/w). Four further animals were treated with 0.05 mL of a 50%, 25%, 10% and 5% (w/w) test item concentration.
- Epidermal application: Test item in corn oil was applied the shaved left flank using a Metalline patch 2x3 cm mounted on Micropore tape and held in place with Coban elastic bandage.
After 24 hours, the dressing was removed and the residual test item removed with a tissue moistened with tap-water. The treated skin areas were assessed for irritation 24 and 48 hours after removal of the bandage. After the 24 hour skin assessment the treated areas where re-shaved.

MAIN STUDY
INDUCTION
- No. of exposures: 9
- Exposure period: 6 hours, after each exposure period residual test item was removed with a tissue moistened with tap-water.
- Test group: test item
- Control group: vehicle
- Site of application: the scapula region (left side), prior to each application the skin was clipped
- Frequency of applications: Days 1, 3, 5, 8, 10, 12, 15, 17, 19
- Duration: 3 weeks
- Concentration: 25% (w/w)
- Method of application: The test item suspended in corn oil was applied to skin under a piece of
metalline (2 x 3 cm) attached to aluminium foil by means of petrolatum, mounted on sleek tape which was kept in place by elastic bandage.

CHALLENGE EXPOSURE
- No. of exposures: 1
- Days after last induction: 10
- Exposure period: 6 hours, after each exposure period residual test item was removed with a tissue moistened with tap-water.
- Test group: test item
- Control group: test item
- Site: Right flank, prior to application a 5 x 5 cm area was clipped and shaved.
- Concentration: 25% (w/w)
- Method of application: In a similair way as in the induction phase.
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressings.
The control animals were treated equally with omission of the test item

RE-CHALLENGE EXPOSURE
- No. of exposures: 1
- Days after first challenge: 7
- Exposure period: 6 hours, after each exposure period residual test item was removed with a tissue moistened with tap-water.
- Test group: test item
- Control group: test item
- Site: contralateral shaved flank, prior to application a 5 x 5 cm area was clipped and shaved.
- Concentration: 25%, 10% and 5% (w/w)
- Method of application: In a similair way as in the induction phase, except that Square chambers, attached to Micropore tape, were used for complete occlusive conditions.
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressings.
The control animals were treated equally with omission of the test item

OTHER OBSERVATIONS
- Mortality: Once daily.
- Clinical signs: Once Daily.
- Body weights: During acclimatisation and at termination of the study.
Challenge controls:
Not performed
Positive control substance(s):
yes
Remarks:
0.5% (w/w) formaldehyde in milli-RO water

Results and discussion

Positive control results:
The sensitivity check with the positive control was perfomed on April 1990.
Clear positive results were observed in the animals after an induction and challenge phase with 1.0% and 0.5% (w/w) formaldehyde in milli-RO respectively. A sensitisation rate of 35% was obtained.
The sensitivity check with formaldehyde indicates that the Himalayan strain of guinea pig is an a
ppropriate animal model for the performance of studies designed to evaluate the sensitising
potential of a substance.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.5%
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
5, 10 and 25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
5, 10 and 25%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
5, 10 and 25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
5, 10 and 25%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
other: to clarify the possibly sensitisation reaction, a re-challenge was carried out
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

INTERPRETATION
The results evident in test animals at the challenge application(s) were compared with the results evident in control animals.
A positive skin reaction would have been considered a sign of sensitisation, provided that such reactions were not observed in the control group.
The readings after the challenge applications were compared to assess the sensitisation rate i.e. the number of sensitised animals in proportion to the total number of animals of the experimental group.


OTHER RESULTS
Results of the primary irritation study: Based on the results, a test item concentration of 25% (w/w) in corn oil was selected for the induction phase and challenge phase respectively, as higher concentrations were too viscous.
Induction phase: No animals showed skin irritation after the last epicutaneous induction exposure.
First challenge phase: Seven animals in the test chemical group showed red spots in response to the 25% test substance concentration in the second reading. To clarify if this was a clear sensitisation reaction, a re-challenge under complete occlusive conditions was performed one week later.


Re-challenge phase: None of the animals showed a skin reaction in response to any of the concentrations tested.



- Mortality / systemic toxicity: No mortallity occurred and no symptoms of systemic toxicity were observed in the animals.
- Bodyweights: A marked difference in the average body weight gain of the test and control group was noted.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In a Buehler-Test, performed according to OECD guideline 406 and GLP, an epicutaneous exposure of 25% (w/w) MDI/CHA caused no sensitization reaction to guinea pig skin in the re-challenge phase (sensitization rate of 0%). Based on these results, the test substance is not classified as skin sensitizing according to Regulation (EC) No 1272/2008 and its amendments.