Registration Dossier

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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
pre guidline study, addresses key parameters of OECD 429
Deviations:
not applicable
Principles of method if other than guideline:
Groups of mice (n = 4) were exposed topically on the dorsum of both ears to 25 gl of various concentrations of the test chemical in 4:1 acetone: olive oil (AOO). Control mice received an equivalent volume of vehicle alone. Three days later all mice were injected intravenously via the tail vein with 20 eCi of [ 3 H]methylthymidine (sp act 2 Ci/mmol; Amersham International, Amersham, Bucks, UK) in 250 "l of phosphate-buffered saline (PBS). Five hours later mice were killed and the draining auricular lymph nodes excised and pooled for each experimental group. A single cell suspension was prepared by gentle mechanical disaggregation through 200mesh stainless steel gauze. LNC were washed twice with an excess of PBS and precipitated in 5% trichloroacetic acid (TCA). Twelve hours later pellets were resuspended with I ml ofTCA and transferred to 10 ml of scintillation fluid (Optiphase MP, LKB, Flow McClean, VA). Incorporation of [3 H]thymidine ( 3HTdR) was measured by *scintillation and results were expressed as the mean cpm per node for each experimental group.
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: 
- Strain: BALB/c - Sex: female
- Source: Barriered Animal Breeding Unit, Alderley Park (UK)
- Age: 8-12 weeks
- Controls: vehicle
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0; 0.05; 0.1; 0.25; 0.5; 0.5; 1.0; 2.5; 0.5% (w/v) / 25 µL
No. of animals per dose:
4
Details on study design:
Groups of mice (n = 4) were exposed topically on the dorsum of both ears to 25 µl of various concentrations of the test chemical in 4:1 acetone: olive oil (AOO). Control mice received an equivalent volume of vehicle alone. Three days later all mice were injected intravenously via the tail vein with 20 eCi of [ 3 H]methylthymidine (sp act 2 Ci/mmol; Amersham International, Amersham, Bucks, UK) in 250 "l of phosphate-buffered saline (PBS). Five hours later mice were killed and the draining auricular lymph nodes excised and pooled for each experimental group. A single cell suspension was prepared by gentle mechanical disaggregation through 200mesh stainless steel gauze. LNC were washed twice with an excess of PBS and precipitated in 5% trichloroacetic acid (TCA). Twelve hours later pellets were resuspended with I ml ofTCA and transferred to 10 ml of scintillation fluid (Optiphase MP, LKB, Flow McClean, VA). Incorporation of [3 H]thymidine ( 3HTdR) was measured by *scintillation and results were expressed as the mean cpm per node for each experimental group
Positive control substance(s):
not specified
Positive control results:
no data
Key result
Parameter:
EC3
Value:
0.073
Key result
Parameter:
SI
Value:
1.81
Test group / Remarks:
0.05 % (w/v)
Parameter:
SI
Value:
4.39
Test group / Remarks:
0.1 % (w/v)
Parameter:
SI
Value:
23.21
Test group / Remarks:
0.25 % (w/v)
Parameter:
SI
Value:
30.58
Test group / Remarks:
0.5 % (w/v)
Parameter:
SI
Value:
40.16
Test group / Remarks:
1.0 % (w/v)
Parameter:
SI
Value:
54.91
Test group / Remarks:
2.5 % (w/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION

SI= B/A

EC3 CALCULATION

EC3= c+[(3-d)/(b-d)](a-c)

CLINICAL OBSERVATIONS and BODY WEIGHTS
Not reported

The study showed an concentration dependend increase of ear thickness relative to pre-challenge values. The optimum response was observed at 1.0% induction concentration. Numeric values regarding ear thickness not published.

Conclusions:
According to the results of this LLNA within this study the test item Isophorone Diisocyanate showed a dermal sensitizing potential.
Executive summary:

In this publication immune responses in mice following topical exposure to three diisocyanates; diphenylmethane-4,4-diisocyanate (MDI), dicyclohexylmethane-4,4-diisocyanate (HMDI), and isophorone diisocyanate (IPDI) were examined.

Lymphocyte proliferative responses in draining lymph nodes were measured 3 days following exposure of mice to various concentrations (0.0; 0.05; 0.1; 0.25; 0.5; 1.0; 2.5 %) of IPDI. IPDI caused a concentration related increase in LNC proliferation. Stimulation indices increase from from 1.81 after treatment with 0.05 % IPDI up to 54.91 after treatment with 2.5%, The EC3 is 0.073 %.

In this mouse ear swelling test performed within this study, mice (6 per dose group) were sensitized via epicutaneous application of 50µl of various concentrations (0.1; 0.25; 0.5; 1.0 and 2.5 %) of the test item IPDI. Five days after sensitization (induction) the challenge was performed by epicutaneous application of 25µl of 0.5% concentration of the test item. Ear thickness was evaluated 24 h after the challenge. The study showed a concentration dependent increase of ear thickness relative to pre-challenge values. The optimum response was observed at 1.0% induction concentration.

These results characterize the test substance Isophorone Diisocyanate as a dermal sensitizer for mice under the conditions of this study.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Method: other: no data
GLP compliance:
not specified
Type of study:
mouse ear swelling test
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: 
- Strain: BALB/c - Sex: female
- Source: Barriered Animal Breeding Unit, Alderley Park (UK)
- Age: 8-12 weeks
- Controls: vehicle
Route:
epicutaneous, open
Vehicle:
acetone/olive oil (4:l v/v)
Concentration / amount:
0.1%; 0.25%; 0.5%; 1.0%; 2.5% (w/v) / 50µL
Route:
epicutaneous, open
Vehicle:
acetone/olive oil (4:l v/v)
Concentration / amount:
0.5% (w/v) / 25 µL
No. of animals per dose:
6
Details on study design:
1st application: Induction open epicutaneous
2nd application: Challenge 0.5 % open epicutaneous
ADMINISTRATION/EXPOSURE 
- Induction schedule: 50 ul on each shaved flank
- Concentrations used for induction:   0; 0.1; 0.25; 0.5; 1.0, 2.5 % w/v
- Challenge schedule: 5 days after induction;   25 ul of 0.5% concentration to the dorsum of both ears
EXAMINATIONS: 
ear thickness before and 24 h after challenge
Challenge controls:
yes; induction concentration 0%; challenge concentration 0.5%
Positive control substance(s):
not specified
Positive control results:
no data
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
induction: 0%; 0.1%; 0.25%; 0.5%; 1.0%; 2.5% (w/v)// challenge: 0.5%
Clinical observations:
see "remarks on results including tables and figures" and "overall remarks"
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: induction: 0%; 0.1%; 0.25%; 0.5%; 1.0%; 2.5% (w/v)// challenge: 0.5%. Clinical observations: see "remarks on results including tables and figures" and "overall remarks".

The study showed an concentration dependend increase of ear thickness relative to pre-challenge values. The optimum response was observed at 1.0% induction concentration. Numeric values regarding ear thickness not published.

Conclusions:
According to the results of this study the test item Isophorone Diisocyanate showed dermal sensitizing potential.
Executive summary:

In this publication immune responses in mice following topical exposure to three diisocyanates; diphenylmethane-4,4-diisocyanate (MDI), dicyclohexylmethane-4,4-diisocyanate (HMDI), and isophorone diisocyanate (IPDI) were examined.

Lymphocyte proliferative responses in draining lymph nodes were measured 3 days following exposure of mice to various concentrations (0.0; 0.05; 0.1; 0.25; 0.5; 1.0; 2.5 %) of IPDI. IPDI caused a concentration related increase in LNC proliferation.

In this mouse ear swelling test performed within this study, mice (6 per dose group) were sensitized via epicutaneous application of 50µl of various concentrations (0.1; 0.25; 0.5; 1.0 and 2.5 %) of the test item IPDI. Five days after sensitization (induction) the challenge was performed by epicutaneous application of 25µl of 0.5% concentration of the test item. Ear thickness was evaluated 24 h after the challenge. The study showed a concentration dependent increase of ear thickness relative to pre-challenge values. The optimum response was observed at 1.0% induction concentration.

These results characterize the test substance Isophorone Diisocyanate as a dermal sensitizer for mice under the conditions of this study.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Cited as Directive 84/449/EEC, B.6
GLP compliance:
not specified
Type of study:
Buehler test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: 
- Strain: Dunkin-Hartley
- Sex: female
- Source: Charles River (France)
- Weight at study initiation: 350 g on average
- Sterile food pellets (UAR 1 14, France) and filtered tap water were supplied ad libitum

ENVIRONMENTAL CONDITIONS:
- room temperature: 18°C (+/-1°C)
- humidity 50% (+/- 5%)
- air ventilation: 15 cycles/h
- 12h light/dark cycle
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
1st application: Induction 5 % occlusive epicutaneous
2nd application: Challenge 1 % occlusive epicutaneous
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
1st application: Induction 5 % occlusive epicutaneous
2nd application: Challenge 1 % occlusive epicutaneous
No. of animals per dose:
20 test / 10 control
Details on study design:
ADMINISTRATION/EXPOSURE 
- Induction schedule: not reported; see guideline
- Concentrations used for induction: 5 % (w/v); 0.5 ml
- Challenge schedule:   14 days after end of induction: patch treatment   approx. 30 hours after patch application: assessment of skin reactions
- Concentrations used for challenge: 1 % (w/v); 0.5 ml
- Positive control: neomycin sulfate (CAS RN 1405-10-3)
- HMDI (CAS: 5124-30-1) was selected as positive reference substance

EXAMINATIONS
- Grading system: Magnusson/Kligman   
0 = no visible change   
1 = discrete or patchy erythema   
2 = moderate and confluent erythema   
3 = intense erythema and swelling   only scores of 2 and/or 3 considered positive;    
histopathological examination in cases of doubt   
Characterization of sensitization potential in 5 groups according to  the number of positive animals
- Pilot study: determination of test concentrations for induction (mild  to moderate dermal response or 100 %) and challenge (no dermal response)
Challenge controls:
Controls: 10 animals; vehicle during induction
Positive control substance(s):
yes
Remarks:
neomycin sulfate (CAS# 1405-10-3)
Positive control results:
sensitization rate Neomycin sulfate: 10/19 (Induction 50% (w/v); challenge 10% (w/v))
sensitization rate HMDI: 19/20 (Induction 2% (w/v); challenge 0.5% (w/v))
Reading:
1st reading
Hours after challenge:
30
Group:
test chemical
Dose level:
1 %
No. with + reactions:
16
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 30.0. Group: test group. Dose level: 1 %. No with. + reactions: 16.0. Total no. in groups: 20.0.
Reading:
1st reading
Group:
test chemical
Dose level:
vehicle
Clinical observations:
vehicle controls did not induce irritation and/or sensitization
Remarks on result:
other: Reading: 1st reading. Group: test group. Dose level: vehicle. Clinical observations: vehicle controls did not induce irritation and/or sensitization.
Conclusions:
Under the conditions of this study the test item induces strong skin sensitization.
Executive summary:

In this study Buehler tests were performed with a series of Diisocyanates, including the test item 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate; IPDI (CAS# 4098-71-9 / EC# 223-861-6), in female Dunkin-Hartley guinea pigs. Neomycin sulfate was used as a positive control to demonstrate the validity of the test.


For the induction 5 % of the test item was applied occlusive epicutaneous, 14 days after induction for the challenge 1 % of the test item was applied occlusive epicutaneous by patch treatment. Approximately 30 hours after the patch application skin reactions were assessed according to the grading system of Magnusson/Kligman:


0 = no visible change   


1 = discrete or patchy erythema   


2 = moderate and confluent erythema   


3 = intense erythema and swelling   only scores of 2 and/or 3 considered positive;    


histopathological examination in cases of doubt   


 


Evaluation of the sensitizing potential of substances is based only on the number of guinea pigs showing positive sensitization (only guinea pigs with a score of 2 and/or 3 were considered as positive in this experiment) and not on the reaction intensity.


In the Buehler test performed within this study, after occlusive epicutaneous induction with 0.5 ml of a solution of 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate in petrolatum at 5% (w/v), 16/20 guinea pigs showed positive response upon occlusive epicutaneous challenge with 1% test substance. This characterizes the test substance as a strong sensitizer under the conditions of this study.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-05-04 to 1983-06-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions: Purity of test substance not reported
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A skin sensitization test according to OECD 406 has already excisted since 1983 and is sufficient for evaluation of the skin sensitisation potential of the test substance.
Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS: 
- Strain: Dunkin-Hartley, Pirbright White, Hoe: DHPK (SPF - LAC.) /Boe.
- Sex: no data
- Source: Lippische Versuchstierzucht Hagemann, Extertal (Germany)
- Weight at study initiation: mean 350 g
- Controls: 20 animals, concurrent vehicle
- 2 Animals per cage in Makrolon III cages
- Sniff Bedding from fir-, spruce-, pine wood, dried; cleared from dust and sterilised
- diet: ssniff G (Ssniff, soest)
- water ad libitum
- Acclimation period: approx. 7 days

ENVIRONMENTAL CONDITIONS:
- room temperature: 21°C (+/- 2°C)
- humidity: 45-55%
- light: 12h/day
Route:
intradermal and epicutaneous
Vehicle:
other: Paraffin oil (DAB 6)
Route:
intradermal
Concentration / amount:
1st application: Induction 10 % intracutaneous in paraffin
10 % intracutaneous in FCA which was diluted 1:1 with Oleum rachaidis prior to mixing with the test item

control group 1:1 FCA in aqua dest
paraffin
Adequacy of induction:
not specified
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:

2nd application: Induction undiluted occlusive epicutaneous

Route:
epicutaneous, occlusive
Vehicle:
other: Paraffin oil (DAB 6)
No.:
#3
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:

3rd application: Challenge undiluted occlusive epicutaneous
No. of animals per dose:
20 test / 20 control
Details on study design:
ADMINISTRATION/EXPOSURE 
- Induction schedule:    
Day 0: Injection   
- Injection details: pairwise injections of 0.05 ml each on shoulders:  
2 x test substance 10 % in vehicle (paraffin)    (control: vehicle)   
2 x test substance 10 % in 50:50 mixture of Freund's Complete Adjuvant (FCA) / oleum arachidis   (control: vehicle instead of test substance)   
2 x FCA / distilled water (50:50)   (control: FCA undiluted) - Days 7-9: 48 hours closed patch treatment of injection sites (0.5 ml;  control: vehicle)
- Challenge schedule:    
Days 21-22: 24 hour closed patch treatment with test substance (left  flank) / vehicle (right flank)   
Days 22-23: Readings at patch removal and 24 hours later
- Concentrations used for challenge: 100 % (0.5 ml)
- Rechallenge: no - Positive control: none

EXAMINATIONS
- Grading system:   
0 = no skin reaction   
0.5 = slight and spotted erythema   
1 = slight and regular, or moderate and spotted erythema   
2 = moderate erythema    
3 = severe erythema or edema

- Pilot study: range finding (skin irritation)   Test substance undiluted; 75 %; 50 % in vehicle tested in 2 animals per  concentration   
Single dermal treatment with 0.5 ml, probably (not reported) 24 hour  occlusive patch  
Observation period 4 days after test substance administration
Challenge controls:
Treatment: vehicle and test item
Positive control substance(s):
no
Positive control results:
no positive control
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
undiluted
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
RESULTS OF PILOT STUDY: no irritation at any concentration
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: undiluted. No with. + reactions: 17.0. Total no. in groups: 20.0. Clinical observations: RESULTS OF PILOT STUDY: no irritation at any concentration .
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
RESULTS OF PILOT STUDY: no irritation at any concentration
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: RESULTS OF PILOT STUDY: no irritation at any concentration .
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
undiluted
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
RESULTS OF PILOT STUDY: no irritation at any concentration
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: undiluted. No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: RESULTS OF PILOT STUDY: no irritation at any concentration .
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
RESULTS OF PILOT STUDY: no irritation at any concentration
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: RESULTS OF PILOT STUDY: no irritation at any concentration .
Group:
positive control
Remarks on result:
other: no positive control tested in this study
Remarks:
validation tests not reported
Conclusions:
Under the conditions of this guinea pig maximization test, the test item 3-Isocyanatomethyl-3,3,3-trimetylcyclohexylisocyanate exhibited the potential to produce dermal sensitization in guinea pigs.
Executive summary:

The clear liquid test item 3 -isocyanatomethyl-3,5,5 -trimethylcyclohexyl isocyanate; IPDI ( CAS# 4098 -71 -9 / EC# 223 -861 -6) was tested in a Guinea pig maximization test (Magnussen and Kligmann) according to OECD 406 for its sensitizing properties.

20 Dunkin-Hartley, Pirbright White guinea pigs with a mean body weight of 350 g were used in this study for both, for the test item group as well as for the control group. Prior to the main study a pilot range finding study in order to assess skin irritation by the test item was conducted. The test item was tested undiluted; 75 % and  50 % in the vehicle paraffin in 2 animals per concentration.

The induction was conducted on day 0 intradermal and after 7 days epicutane:

Day 0: Pairwise injections of 0.05 ml each on shoulders:  

-         2 x test substance 10 % in vehicle (paraffin) (control: vehicle)   

-         2 x test substance 10 % in 50:50 mixture of  FCA / oleum arachidis

(control: vehicle instead of test substance)   

-         2 x FCA / distilled water (50:50)   (control: FCA undiluted)       

Day 7: 48 hours closed patch treatment of injection sites (0.5 ml undiluted test item; control: vehicle)

The challenge was conducted after 21 -22 days for 24 hour by closed patch treatment of the undiluted the test item (0.5 mL; left flank) / vehicle (right flank) and readings were conducted at patch removal and 24 hours later.

 

Grading system:   

- 0 = no skin reaction   

- 0.5 = slight and spotted erythema   

- 1 = slight and regular, or moderate and spotted erythema   

- 2 = moderate erythema    

- 3 = severe erythema or edema

 

The test item showed no indication of primary irritation in the range finding study in concentrations up to 100%.

24 hours after the challenge, 17 out of 20 animals were positive having an overall mean score of 1.15 (max.3). After 48 hours 16 out of 20 animals were positive having an overall mean score of 0.85 (max.3). 24 and 48 hours after the challenge 19 out of 20 animals showed a positive reaction. In the control group 0 out of 20 animals were positive at 24 and at 48 hours after the challenge.

Under the conditions of this guinea pig maximization test, the test item 3 -Isocyanatomethyl-3,3,3 -trimetylcyclohexylisocyanate exhibited the potential to produce dermal sensitization in guinea pigs. These results characterize the test substance as a dermal sensitizer under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Partly cited from SIAR for SIAM 23 (Jeju, Korea, October 17-20, 2006):


Studies in Animals


3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate was found to be sensitizing in numerous studies. Positive results were obtained in the Buehler test performed according or equivalent to the corresponding EU Directive (Zissu, Binet and Limasset, 1998;American Cyanamid Company, 1987), in the guinea pig maximization test comparable or according to OECD TG 406 (IBR/Huels AG 1983, Schmidt/Bayer AG, 1984; Vohr, 1993), in the mouse ear swelling test (Dearman, Spence and Kimber, 1992), and in the open epicutaneous test (Biosphere Research Center Inc., 1981).


"For example, in the Buehler test performed by Zissu, Binet and Limasset (1998), after occlusive epicutaneous induction with 0.5 ml of a solution of 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate in petrolatum at 5% (w/v), 16/20 guinea pigs showed positive response upon occlusive epicutaneous challenge with 1% test substance. This characterizes the test substance as a strong sensitizer. Similarly, in the Guinea pig maximization test performed by Vohr (1993) using 0.1 ml of a 5% solution in olive oil for intracutaneous induction, 15/20 guinea pigs from the test group displayed a positive response upon semiocclusive rechallenge at 0.1%. However, in this study skin reactions were also observed in control animals, though at a lower incidence as compared to the test group, which is why a second challenge was performed."


 


 


Studies in Humans


"A glue, mainly based on dicyclohexylmethane-4,4'-diisocyanate (70%), was suspected of being the cause of an outbreak of severe eczema at a factory manufacturing medical equipment from August 1999 to April 2001 (Frick et al., 2003). 16 out of approximately 100 persons working in the relevant department were referred to medical consultation. When patch tested with a standard series, an isocyanate series, and work material, 4 of these 16 persons reacted to 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate.


Two Italian women who worked with polyurethane materials made of diisocyanates other than 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (diphenylmethane-4,4'-diisocyanate in one case,dicyclohexylmethane-4,4'-diisocyanatein the other) developed distinct contact dermatitis. When patch testedwith the North American Contact Dermatitis Group (NACDG) standard series and with a second series including in one case 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (1% in petrolatum), a weakly positive response towards 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate was observed beside positive responses to other isocyanate materials (Militello et al., 2004).


Twenty poorly documented cases of occupational dermatoses observed between the end of 1970 and mid 1974 were reported in East Germany (Rothe, 1976). Appropriate concentrations for patch epicutaneous challenge testing were determined by self-application of medical staff. 1% solutions of 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate in acetone as well as test solutions of other isocyanates were then applied to workers who were suspected to be sensitized by polyurethane chemicals. Readings were done at 24, 48, and 72 hours (some also at 96 hours). Four persons turned out to be sensitized towards 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate. The main symptoms in these cases were follicular nodules. Symptoms had appeared after an accidental spill with 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate even in two of the above mentioned persons that had previously no contact with this substance, but with toluene diisocyanate and diphenylmethane diisocyanate. The skin of the sensitized workers returned to a stable healthy state after avoiding contact with 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate.


In the same poorly documented study, single-dose self-application of medical staff with undiluted 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate caused follicular papules after 10 days in 2 out of 3 persons. Sensitization was confirmed by challenge with 1% 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate in acetone. Control tests in 6 non-exposed persons with eczema were negative (Rothe, 1976).


Cross-sensitivity between 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate and the corresponding diamine 3-aminomethyl-3,5,5-trimethylcyclohexylamine was studied by Lachapelle and Lachapelle-Ketelaer (1979). Two workers who were allergic to the diamine and two volunteers who had been sensitized also to the diamine were patch tested 1 month later with 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (1% in ethanol); the patches were removed after 48 hours, and read at 48 and 96 hours. Five adult volunteers were patch tested with 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate as controls. The tests were strongly positive in the 4 patients. None of the control subjects was positive." Since 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate is hydrolyzed (see chapter 2.2.3 of SIAR), which initially leads to the diamine (see chapter 3.1.1 of SIAR), traces of 3-aminomethyl-3,5,5-trimethylcyclohexylamine are expected to occur in patch tests with 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate and may trigger symptoms of sensitization in persons who are allergic towards 3‑aminomethyl-3,5,5-trimethylcyclohexylamine.


"Non-occupational skin sensitization towards 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate was identified by Belsito (2003). Three out of 70 patients with allergic-appearing foot dermatitis, of which 23 were found to have allergic contact dermatitits from shoes, showed positive response when challenged with 1% 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate in petrolatum. The source of exposure appeared to be the foam rubber padding in athletic shoes, though migration from glues into the padding could not be excluded."


Liippo and Lammintausta (2008) describe patch testing with isophorone diisocyanate in 433 dermatology patients. 8 patients from 433 tested patients showed positive reactions (1.8% of the tested patients). Cross reactivity between isophorone diamine (IPDA) and isophorone diisocyanate was apparent for two of the patients. In general according to the investigated patients the association with current dermatitis was not apparent in all cases.



Short description of key information:
Cited from SIAR for SIAM 23 (Jeju, Korea, October 17-20, 2006):
"3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate was found to be skin sensitizing in the Buehler test according to or equivalent to the EU Directive, in the guinea pig maximization test comparable or according to OECD TG 406, in the mouse ear swelling test, and in the open epicutaneous test. Skin sensitization was also observed in humans."

Respiratory sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-01-31 to 1994-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
other: exposure criteria of OECD Guideline 403 (1981) and Directive 84/449/EEC, B.6 fulfilled
Deviations:
not applicable
Principles of method if other than guideline:
exposure criteria of OECD Guideline 403 (1981) and Directive 84/449/EEC, B.6 fulfilled
Pauluhn J and Eben A (1991). Validation of a non-invasive technique to  assess immediate- or delayed-onset of airway hyperreactivity in  guinea-pigs.
 J. Appl. Toxicol. 11, 423-431; Pauluhn J (1994). Validation of an improved nose-only exposure system for  rodents. J. Appl. Toxicol. 14, 55-62.
GLP compliance:
yes
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS: 
- Strain: Hartley [Crl:(HA)BR]
- Sex: female
- Source: Charles River Wiga, Sulzbach (Germany)
- Age: ca. 2 months
- Weight at study initiation: 237-273 g; mean 256 g
- pre-experimental treatment of animals and animal housing: animals were acclimatized for at least 5 days before use; before start of experiment health status of each animal was assessed; animals were assigned to exposure gruops at random;
- diet: Altromin 3022 (Altromin GmbH, Lage)
- tap-water ad libitum

ENVIRONMENTAL CONDITIONS:
- room temperature: 22 °C (+/- 2°C)
- humidity: appr. 50%
- dark/light: 12h/12h (14 watt/m2)
- air-exchange: appr. 10-fold/h
Route of induction exposure:
intradermal
Route of challenge exposure:
other: nose only inhalation of aerosol
Vehicle:
other: corn oil
Concentration:
first day: 10.2 mg/m3 test substance 30 min nose-only   
second day: 0.05; 0.15; 0.5 % Acetylcholine for 15 min each   
last day: 35.5 mg/m3 conjugate of test substance with guinea pig serum  albumin
No. of animals per dose:
8 treated
8 vehicle (control)
Details on study design:
1st application: Induction 0.1 % intracutaneous
2nd application: Challenge other: nose only inhalation of aerosol
ADMINISTRATION/EXPOSURE 
- Induction schedule: days 0, 2, 4 (100 ul each)
- Challenge schedule: 4 subgroups, 4 animals each   
days 21/22/28 (control subgroup a)   
days 22/23/29 (treated subgroup a)   
days 23/24/30 (control subgroup b)   
days 24/25/31 (treated subgroup b)
- Concentrations used for challenge:    
first day: 10.2 mg/m3 test substance 30 min nose-only   
second day: 0.05; 0.15; 0.5 % Acetylcholine for 15 min each   
last day: 35.5 mg/m3 conjugate of test substance with guinea pig serum  albumin
EXAMINATIONS
- During sacrifice the trachea, lung and lung associated lymph nodes were  fixed and subjected to histopathological evaluation.  Lung weights were  also determined - Pilot study: assessment of the approximate irritant threshold  concentration
Challenge controls:
Validity of this animal model was assessed with the known human sensitizer Trimellitic Anhydride. Additional ACh challenge was performed to enhance identification of and distinction between specific as well as non specific airway hyperresponsiveness.
Positive control substance(s):
trimellitic anhydride (TMA)
Negative control substance(s):
other: vehicle (corn oil)
Results:
RESULTS OF PILOT STUDY: see test concentration RESULTS OF TEST - Sensitization reaction: High titer IgG1 antibody observed proved that  successful sensitization had occurred. However, 
when challenged, the  incidence of immediate-onset respiratory reactions was roughly the same  in all groups. No delayed-onset 
reactions, deaths or anaphylactic  reactions were observed. Challenge with acetylcholine did not evoke group  specific respiratory 
responses. - Clinical signs: No clinical signs or specific abnormalities were  observed at necropsy.
Positive control results:
Severe reactions were observed with trimellitic anhydride (CAS RN  552-30-7) when investigated with the current animal model, 
using the same  induction and challenged.
Negative control results:
no relevant reactions observed

no further remarks

Conclusions:
Under the conditions of this study the test item did not induce respiratory sensitization in guinea-pigs.
Executive summary:

Respiratory tract sensitization of guinea pigs following intradermal induction (1%, 100 µl) was examined in this study in accordance with the exposure criteria defined in OECD TG 403. High titer IgG1 antibody observed proved that successful sensitization had occurred. However, when challenged by nose only inhalation of aerosol at varying concentrations, the incidence of immediate-onset respiratory reactions was roughly the same in all groups. No delayed-onset reactions, deaths or anaphylactic reactions were observed. Challenge with acetylcholine did not show specific respiratory responses indicating that the animals were hyperrespondive to cholinergic acetylcholine stimuli. Severe reactions were observed with trimellitic anhydride (CAS No. 552-30-7) when investigated with the current animal model, using the equivalent induction and challenge. Under the conditions of this study the test item did not induce respiratory sensitization in guinea-pigs.

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study without detailed documentation but in good agreement with generally accepted scientific principles.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Modified LLNA test using the inhalation route followed by an assessment of cytokine profiles (IL-4, IL-10, IL-12, IFN-gamma) in the draining lymph nodes
GLP compliance:
not specified
Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals or test system and environmental conditions:
Test organism:
- BALB/c mice (inbred)
-Sex: male
-age: 6-7 weeks
Source: Charles River Deutschland (Sulzfeld, Germany)
Environmental conditions:
-animals were bred under specific pathogen free conditions (SPF)
- housed (3 or 6 per group in macrolon cages) under conventional conditions in light-, humidity- and temperature-controlled rooms
-diet: standard pellet diet (RM3 SQC, Special Diet Service, Witham, UK)
-water: unfuorinated tap water ad libitum
Route of induction exposure:
inhalation
Remarks:
head/nose only
Route of challenge exposure:
other: not described
Vehicle:
other: Acetone (inhalation exposure); AOO (dermal exposure)
Concentration:
7.5 mg/m3
No. of animals per dose:
IPDI:
-6 animals for each group
Vehicle:
6 or 12 animals for vehicle control group
Details on study design:
Respiratory LLNA:
-6 or 12 animals for vehicle control group
-mice were exposed to fixed concentration (7.5 mg/m3) via inhalation (head/nose only)
- Groups (6 animals per group) were exposed for 45, 90, 180 or 360 min on 3 consecutive days (day 0, 1, 2)
- vehicle control group (0.06% acetone in air; 6 or 12 animals per group) were exposed for 360 min/day
-day 5: lymph nodes were collected and used for ex vivo cell proliferation and cytokine measurement and respiratory tract was examined microscopically

Skin LLNA:
-skin LLNA served as control in order to be compared with effects found in respiratory LLNA
- 25 µl IPDI dissolved in acetone:olive oil (4:1) solution (AOO) was applied to the dorsum of both ears for 3 consecutive days (days 0,1,2)
- Control (n=6 animals): AOO only
- IPDI (3 animals): 1%
- day 5: auricular lymph nodes were collected and used for ex vivo cell proliferation and cytokine measurement and respiratory tract was examined microscopically

Collection of lymph nodes (LN):
- mandibular LNs were taken out
-only were grossly observed enlargement of other LNs was noted these LNs were collected additionally (e.g. posterior cervical and auricular LNs in case of IPDI exposure)

Preparation of single cell suspensions and assessment of cytokine production:
- see: description in Arts et al., 2008
Challenge controls:
No direct challenge control. Positive reaction after skin exposure to known sensitizers (trimellitic anhydride (TMA); phthalic anhydride (PA); toluene-diisocyanate (TDI), hexamethylene-1,6-diisocyanate(HDI); isophorone diisocyanate (IPDI); 2,4-dinitrochlorbenzene (DNCB); oxazolone (OXA); formaldehyde (FA)) severed as control. Negative control was the irritant methyl salicylate .
Positive control substance(s):
other: exposure via inhalation: skin application served as positive control: trimellitic anhydride (TMA); phthalic anhydride (PA); toluene-diisocyanate (TDI), hexamethylene-1,6-diisocyanate(HDI); isophorone diisocyanate (IPDI); 2,4-dinitrochlorbenzene (DNCB); o
Negative control substance(s):
other: exposure via inhalation: skin application served as negative control: methyl salicylate (MS) and vehicle control
Results:
Local effects, body weights and necropsy after inhalation exposure:
- inhalation exposure relatively well tolerated and no abnormalities were noted during the exposure
- Clinical signs after exposure with IPDI: blepharospasm
- Body weight after exposure with IPDI: slight to moderate losses between day 0 and day 5 in 360 min exposure group only
- Necropsy of animals exposed with IPDI: leanness, stained lungs

Local lymph node activation:
- mandibular lymph nodes in animals exposed with IPDI via inhalation: statistically significant increases in SI > 3 (90, 180 and 360 min/day); dos e response relationship exists
- auricular lymph nodes in animals exposed with IPDI via inhalation: statistically significant dose related increases in SI > 30 (90, 180 and 360 min/day)

Airway histopathology after exposure via inhalation:
- IPDI: moderate lesions in nasal tissues (6/6), exudative rhinitis (3/6)

Cell proliferation after inhalative exposure:
- respiratory sensitizers TMA, PA, TDI, HDI, IPDI: induced positive proliferation (SI >3) in mandibular LN
- contact sensitizers DNCB, OXA: induced positive proliferation (SI >3) in mandibular LN
- contact sensitizer FA and the irritant MS: proliferation not induced (SI < 3) in mandibular LN
-inhalation exposure to TDI, HDI, IPDI and Oxa stimulated auricular LNs as well inducing higher SI values than in the mandibular LNs

IL-4 induction after inhalative exposure:
-respiratory sensitizers TMA, PA, TDI, HDI, IPDI: positive IL-4 response induced
-contact sensitizers DNCB, OXA: positive IL-4 response induced
- FA und MS did not induce positive IL-4 respond
-IPDI induced a linear dose-response curve
-in general respiratory sensitizers induce more IL-4 than contact sensitizers (e.g. IPDI: 2-fold higher than DNCB)

IL-4 induction after skin exposure:
-all respiratory sensitizers induced considerably higher IL-4 responses in the auricular LNs when compared to the contact sensitizers DNCB, OXA and FA
- MS (irritant) did not induce measureable IL-4 response

IFN-gamma induction after inhalative exposure:
-highest induction was found for contact sensitizers (DNCB and OXA)
-DNCB induced approximately 10-fold higher levels of IFN-gamma than IPDI
-no dose-response was found after exposure to IPDI

IFN-gamma induction after skin exposure:
-after skin exposure IFN-gamma was induced by both the respiratory sensitizers and the contact sensitizers (DNCB; OXA)
-IPDI induced lower levels of IFN-gamma compared to HDI, TDI, DNCB and TMA
-

IL-4 as a function of the prolifeative response
-at intermediate SI values (4-7) respiratory sensitizers (except HDI) induced higher levels of IL-4 than the contact sensitizers
-compared to DNCB (at SI values: 4-7) IL-4 level was 2.5-fold higher for IPDI (p=0.001)
-compared to OXA (at SI values 4-7) IL-4 level was 3.1-fold higher for IPDI (p=0.0005)
Positive control results:
Positive skin exposure control identified all investigated sensitizers (trimellitic anhydride (TMA); phthalic anhydride (PA); toluene-diisocyanate (TDI), hexamethylene-1,6-diisocyanate(HDI); isophorone diisocyanate (IPDI); 2,4-dinitrochlorbenzene (DNCB); oxazolone (OXA); formaldehyde (FA);
-positive result regarding cell proliferation ( all SI>3).
Negative control results:
Cell proliferation reaction negative after skin exposure for the irritant methyl salicylate (MS). Vehicle controls negative (SI<3).

furthermore IL-10 induction and IL-12 induction was examined after inhalation and skin exposure. Because of lower relevance these results are not described here.

Conclusions:
It was shown that after inhalation exposure to respiratory sensitizers relatively high levels of IL-4 and low levels of IFN-gamma were produced than after inhalation exposure to contact sensitizers. Thus, after inhalation exposure induction of IL-4 could be used to identify respiratory sensitizers both at intermediate and high SI values. Regarding to the results of this study the test item Isophorone diisocyanate (IPDI) has to be considered to have respiratory sensitizing potential.
Executive summary:

In a LLNA test using inhalation instead of skin exposure, a selection of contact and respiratory sensitizers was investigated. Contact and respiratory sensitizers were identified in a respiratory LLNA test by measuring an increase in lymphocyte proliferation in the mandibular lymph nodes. In addition, assessment of cytokines provided information that enabled discriminiation between respiratory and contact sensitizers. It was shown that after inhalation exposure to respiratory sensitizers relatively high levels of IL-4 and low levels of IFN-gamma were produced than after inhalation exposure to contact sensitizers. Thus, after inhalation exposure induction of IL-4 could be used to identify respiratory sensitizers both at intermediate and high SI values.

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study without detailed documentation but in good agreement with generally accepted scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
Dermal exposure of mice to isocyanates including an intranasal challenge by instillation of the isocyanates followed by examinations of respirative responsiveness, bronchoalveolar lavage fluid (BALF), serum and cytokine message in the parotid lymph nodes.
GLP compliance:
not specified
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals or test system and environmental conditions:
Age of animals: 8 weeks
source: Charles River Breeding Laboraties (Raleigh, NC, USA)
feeding: Prolab RMH 3000 (PMI Nutrition International, St. Louis, MO, USA)
water: ad libitum
light/dark cycle: 12h light/12 hours dark
room temerature: approximately 22°C
humidity: approximately 50%

Route of induction exposure:
dermal
Remarks:
back and ear
Route of challenge exposure:
intranasal
Remarks:
intranasal instillation
Vehicle:
other: dermal exposure: Acetone:olive oil(4:1, v/v); challenge by intranasal instillation: ethyl acetate:olive oil (1:4); Metacholin induced response: air and saline
Concentration:
Experiment A: IPDI (2%)
Experiment B: IPDI (2%)
Experiment C: IPDI (2%)
No. of animals per dose:
Experiment A,B,C: 6 mice per group in each experiment
Details on study design:
Experiment A:
- Day 0 and day 5: 100 µl IPDI applied to shaved back ; vehicle control grou similar treated
- Days 10, 11, 12: 25 µl IPDI applied to each side of both ears; vehicle control group similar treated
- Day 14: mice sacrified and parotid lymph nodes removed, cell suspensions were made ana analyzed for cytokine profiling (IL-4, IL-5, IL-13 and IFN- gamma); blood was collected from these mice

Experiment B:
- Day 0 and day 5: 100 µl IPDI applied to shaved back; vehicle control group similar treated
- Days 10, 11, 12: 25 µl IPDI applied to each side of both ears; vehicle control group similar treated
- Day 19: 100µl IPDI applied to shaved back; vehicle control group similar treated
- Day 24: intranasal challenge by instillation with 60 ml (?) (maybe a mistake in the publication, 60 µl is more likely) of a 1% solution of IPDI in 1:4 ethyl acetate and olive oil (lightly anesthetized using 3% isoflurane); vehicle control group similar treated; assessment of immediate airway hypersensitivit y immediately after instillation
- Day 26: mice assessed for Metacholine (Mch) responsiveness; mice sacrified and bronchoalveolar lavage fluid (BALF) and blood collected

Experiment C (control for nonspecific responses):
- Day 0 and day 5: 100 µl vehicle applied to shaved back
- Days 10, 11, 12: 25 µl vehicle applied to each side of both ears
- Day 19: 100µl vehicle applied to shaved back
- Day 24: intranasal challenge by instillation with 60 ml (?) (maybe a mistake in the publication, 60 µl is more likely) of a 1% solution of IPDI in 1:4 ethyl acetate and olive oil (lightly anesthetized using 3% isoflurane); assessment of immediate airway hypersensitivity immediately after instillation
- Day 26: mice assessed for Metacholine (Mch) responsiveness; mice sacrified


Draining lymph node cell suspensions and cytokine response (experiment A):
- parotid lymph nodes from mice of experiment A were excised
-cell suspensions were made, cells were cultured for 24 h (in presence of 2 mg/ml concanavalin ConA) and supernatants were collected and examined regarding the following cytokines IL-4, IL-5, IL-13, IFN-gamma as described in Farraj et al. 2007

In vivo airway responses:
-antigen specific airway responsiveness was measured on day 24 immediately after exposure in unrestrained mice in whole body plethysmography (Buxco Electronics, Troy,NY, USA)
-nonspecific airway responsiveness to increasing concentrations (10, 32,100 mg/ml) of aerosolized Methacholine (Mch) was assessed in unanesthetized unrestrained mice in a 12-chamber whole body plethysmograph system (Buxco Electronics, Sharon, CT) 48 h after intranasal challenge on day 26
-in general enhanced pause (Penh) was calculated which represents the parameter for airway response

Serum collection and bronchoalveolar lavage (experiment B):
- mice anesthetized by i.p. injection of 0.5 ml sodium pentobarbital (5 mg/ml)
- blood sampling by cardiac puncture
- serum collected and stored at -80°C
- Bronchoalveolar lavage: cannula inserted into the trachea; lungs were lavaged twice with a aliquot of 1 ml HBSS (CA/Mg and phenol red free), samples were stored on ice until centrifuged at 360xg for 10 min at 4°C; cell pellet was resuspended in 1 ml HBSS. Stained slides were made from the cells. Differetial cell counts were performed

Histopathology (experiment B):
- histopathologically examined: left lung lobe, nasal cavity (proximal, middle and distal section); tissues sectioned at 5µm and stained with hematoxylin/eosin

Total serum IgE (experiment B):
- measured in mice 48 h after intranasal challenge; performed as described in the materials and methods chapter of Farraj et al.(2007)

statistics:
-data analyzed using analysis of variance (ANOVA); level of significance for evaluation of factors or paiwise comparisons was set at p<0.05
Challenge controls:
Yes: described in section "details on study design"
Positive control substance(s):
other: not applicable; no positive controls used
Negative control substance(s):
2,4-dinitrochlorobenzene (DNCB)
other: vehicle
Results:
Experiment A:
Cytokine response (increase) from lymph node cells stimulated ex vivo with ConA from mice that were topically exposed to the examined substances (expressed assignificant x-fold increase relative to mice exposed to vehicle):
- IL-4: HMDI (181-fold); TDI (157-fold); MDI (72-fold); IPDI (58-fold); TMI (8-fold); TMXDI (3-fold); no significant differences between mice exposed to DNCB and mice exposed to vehicle
- IL-5: HMDI (11-fold); MDI (27-fold); TDI (29-fold); no significant differences between mice that were exposed to IPDI, TMI, TMXDI or DNCB and mice exposed to vehicle
- IL-13: HMDI (55-fold); TDI (82-fold); MDI (63-fold); IPDI (26-fold); TMI (7-fold); TMXDI (5-fold); DNCB (3-fold)
-IFN-gamma: HMDI (43-fold); TDI (88-fold); MDI (340-fold); IPDI (50-fold); TMI (12-fold); TMXDI (6-fold); DNCB (8-fold)

Airway physiology (experiments B and C):
- all substances (except TMXDI) showed significant increases in average Penh immediately after intranasal chemical challenge of sensitized mice relative to both vehicle-exposed and unsensitized mice challenged with the substance (control for nonspecific responses)
- after treatment with metacholine (Mch): very small differences between chemical treatment groups in response to Mch at baseline and Mch concentrations of 10 and 32 mg/ml; after sensitization and challenge with the examined substances followed by treatment with 100 mg/ml Mch: HMDI (1.8 fold) and TMI (1.9-fold) increase in Penh relative to vehicle-challenged mice; dermal sensitization and intranasal challenge with IPDI, MDI, TDI or DNCB did not result in any significant differences relative to the vehicle controls nor were there differences from vehicle in any of the unsensitized groups challenged with the examined substances intranasally

Airway pathology (experiments B and C):
Mice dermally sensitized and intranasally challenged with TDI, MDI, HMDI, IPDI; TMI, TMXDI or DNCB:
-moderate sloughing of airway epithelium in septum, naso- and maxilloturbinates and lateral walls in nasal cavity region
- individual cell necrosis in olfactory epithelium of dorsal meatus and ethmoid turbinates
- in general these lesions were likely not an immune-mediated response but rather an irritant effect of the examined substances
- no exposure related alterations were microscopically evident in the lungs of any of the exposed mice.

Total serum IgE (experiments A, B and C):
Experiment A:
- significant increases in total serum IgE in mice treated with IPDI, HMDI, MDI, TDI, TMI, TMXDI or DNCB relative to vehicle control
- response to IPDI was significantly greater than all other groups
Experiments B and C:
- significant increases in total serum IgE after dermal sensitization and intranasal challenge with the following substances relative to vehicle control (= unsensitized mice): HMDI (59-fold); MDI (49-fold); IPDI (33.4-fold), TDI (24.4-fold); DNCB (15-fold); TMI (8.5-fold); TMXDI (5.5-fold)
Positive control results:
not applicable
Negative control results:
Cytokine responses:
- IL-4 significantly greater in all treated groups than in vehicle and DNCB group; vehicle and DNCB groups not significantly different from each other; IL-5 no significant differences between mice that were exposed to IPDI, TMI, TMXDI or DNCB and mice exposed to vehicle; IL-13 significantly greater in all treated groups (including DNCB) than in vehicle and 3-fold enhanced in DNCB treated mice compared to vehicle; IFN-gamma significantly greater in all treated groups (including DNCB) than in vehicle and 8-fold enhanced in DNCB treated mice compared to vehicle

Airway physiology: see detailed description in section "Results"

Airway pathology:
- no lesions in the nasal airways of the vehicle-treated mice
- alterations in DNCB treated mice described in section "Results"
- alterations in mice sensitized with vehicle and challenged with the examined substances lead to similar airway lesions as described in section "Results" for the examined substances

Total serum IgE:
- IgE significantly greater in all treated groups (including DNCB) than in vehicle
- IgE 15-fold enhanced in DNCB treated mice compared to vehicle in experiments B and C

Summary of the results:

Th2 cytokine response:
TDI (strong IL-4, 5, 13); MDI (strong IL-4, 5, 13); HMDI (strong IL-4, 5, 13); IPDI (strong IL-4, 13); TMI (weak IL-4, 13); TMXDI (weak IL-4, 13); DNCB (weak IL-13)

Serum IgE (26 days):                     
TDI (strong); MDI (strong); HMDI (strong); IPDI (strong); TMI (weak); TMXDI (weak); DNCB (weak)

Immediate airway response:     
TDI (yes); MDI (yes); HMDI (yes); IPDI (yes); TMI (yes); TMXDI (no); DNCB (yes)

Nonspecific airway response:
TDI (no); MDI (no); HMDI (yes); IPDI (no); TMI (yes); TMXDI (yes); DNCB (no)

Conclusions:
The purpose of this study was to determine if the cytokine profile induced after dermal sensitization with isocyanates and serum IgE predict immediate and metacholine-induced late respiratory hypersensitivity responses after intranasal challenge. The discordance between dermal cytokine profiles and respiratory responses suggest that dermal responses do not necessarily predict respiratory responses. Serum IgE also was not predictive of respiratory responses to the isocyanates, suggesting that other unknown mechanisms may be involved. The authors conclude, that all the chemicals tested had the capacity to induce immediate airway hypersensitivity responses and/or enhanced responses to metacholine challenge. Additional work is needed to assess the potential of the examined chemicals to induce asthma.
Executive summary:

The purpose of this study was to determine if the cytokine profile induced after dermal sensitization with isocyanates and serum IgE predict immediate and metacholine-induced late respiratory hypersensitivity responses after intranasal challenge. The discordance between dermal cytokine profiles and respiratory responses suggest that dermal responses do not necessarily predict respiratory responses. Serum IgE also was not predictive of respiratory responses to the isocyanates, suggesting that other unknown mechanisms may be involved. Thus, the ex vivo cytokine and serum IgE endpoints do not necessarily predict immediate antigen-specific airway reactivity. Furthermore the observed respiratory responses could not be correlated with differences in nasal or pulmonary airway pathology. Dermal sensitization followed by intranasal challenge with any of the chemicals used in this study did not elicit any pulmonary airway lesions characteristic of occupational allergic airway disease. The authors state, that all the chemicals tested had the capacity to induce immediate airway hypersensitivity responses and/or enhanced responses to metacholine challenge. Additional work is needed to assess the potential of the examined chemicals to induce asthma. An easily measurable biomarker of exposure that universally predicts the respiratory sensitization potential of all chemicals remains elusive. The data suggest that more than one immunologic mechanism can result in the respiratory responses of interest.

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study without detailed documentation but in good agreement with generally accepted scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
method as described in the publication; Cytokine induction in the draining lymph node
GLP compliance:
not specified
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS: 
- Strain: BALB/c
- Sex: female
- Source: Charles River Breeding Laboratories (Raleigh, NC, USA or Kingston, NY, USA)
- Age: 8-12 weeks
- Number of animals: 5-6 per group

ENVIRONMENTAL CONDITIONS
- room temperature: 20°C (+/-1°C)
- humidity: 50% (+/-10%)
- light/dark Cycle: 12h each
Route of induction exposure:
dermal
Route of challenge exposure:
other: epicutaneous
Vehicle:
other: aceton:olive oil (4:1 v/v)
Concentration:
1st application: Induction 2 % open epicutaneous
2nd application: Induction 2 % open epicutaneous
No. of animals per dose:
5-6
Details on study design:
ADMINISTRATION/EXPOSURE 
- Study type: RPA (ribonuclease protection assay) analysis
- Induction schedule:    Days 0 and 5: Dermal application of 100 µl chemical on both flanks   
Days 10, 11 and 12: Dermal application of 12.5 µl chemical to each side  of both ears 
- Concentrations used for induction:   (1) TDI: 1 %   (2) MDI: 2 %   (3) TMI: 1 %   (4) TMXDI: 1 %   (5) HMDI: 2 %    (6) IPDI: 2 %
- Challenge schedule: On day 14 animals were euthanized, auricular lymph  nodes were removed and total RNA was extracted from these tissues 
and  subject to RPA analysis using commercial assay systems.
- Positive control: thought to be included in set of test substances
EXAMINATIONS
- Grading system: not yet established
- Satellite study: Local lymph node assay (LLNA), in part conducted with  female CBA/JHsd mice (7-12 weeks old) from 
Harlan Sprague-Dawley  
(Frederick, MD, USA), done for four of the six test substances:  
(2) MDI: BALB/c; 0.02 / 0.2 or 2 %   
(3) TMI: CBA/JHsd; 0.25 / 0.5 / 1 %   
(4) TMXDI: CBA/JHsd; 0.25 / 0.5 / 1 %   
(6) IPDI: BALB/c; 0.02 / 0.2 or 2 %   
The purpose was to determine whether the doses chosen induced similar  levels of proliferation, thus suggesting immunological equivalence 
and  allowing direct comparison of chemicals in the RPA main test.
- Follow-up study: RPA analysis as described above for two substances:  (3) TMI, (4) TMXDI
Challenge controls:
Concurrent vehicle control. 
Positive control substance(s):
other: within the experiment toluene diisocyanate (TDI) was tested, which could be regarded as positive substance
Negative control substance(s):
2,4-dinitrochlorobenzene (DNCB)
Results:
General: Results are reported only graphically. Main study:    As expected, (1) TDI and (2) MDI induced message for the Th2 cytokines  IL-4, IL-10 and IL-13. 
However, the mRNA for these cytokines was also  elevated in response to the other four test substances. Based on the  
magnitude of the response, the authors differentiated into two distinct  groups:   High responders: (1) TDI, (2) MDI, (5) HMDI   Low responders: (3) TMI, (4) TMXDI, (6) IPDI   There were no statistically significant changes in IL-2, IL-3, IL-5,  IL-9, IL-15, and IFN-gamma relative to vehicle controls. Satellite LLNA: The doses used in the main study were immunologically  equivalent based on similar SI. Follow-up study: Levels of IL-4, IL-10 and IL-13 were higher for both (3)  TMI, (4) TMXDI than for DNCB, though not 
statistically significant for  IL-4 in the case of (4) TMXDI.

Positive control results:
As expected TDI induced message for the Th2 cytokines  IL-4, IL-10 and IL-13. 
Negative control results:
As expected cytokine levels after exposure with DNCB was much weeker than of animals exposed to the test items of this study.

no further remarks

Conclusions:
Biological relevance of this results are still under discussion. Further research is necessary. The authors conclude that the test item Isophorone Diisocyanate has at least respiratory sensitizing potential under the conditions of the study.
Executive summary:

This study is based on the hypothesis on the idea that respiratory sensitizers can be identified based on relativly high expression of cytokines characteristic of Th2 cells. Thus, cytokine profiling may be an effective way to detect respiratory sensitizers. After exposure with the stest item Isophorone Diisocyanate auricular lymph nodes were removed from the animals, the total RNA was extracted and Cytokine mRNA was analysed. Response after exposure with the test item Isophorone Diisocyanate was variable but was comparatively weaker as for strong respiratory sensitizers used in this study. Biological relevance of this results are still under discussion but in general as the result of this study the authors conclude that the test item Isophorone Diisocyanate has at least respiratory sensitizing potential under the conditions of the study.

Endpoint:
respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study without detailed documentation but in good agreement with generally accepted scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
Dermal exposure of mice to isocyanates followed by examinations of respirative responsiveness, bronchoalveolar lavage fluid (BALF), serum and cytokine message in the auricular lymph nodes.
GLP compliance:
not specified
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals or test system and environmental conditions:
Age of animals: 8-12 weeks
source: Charles River Breeding Laboraties (Raleigh, NC, or for LLNA: Kingston NY, USA)
feeding: standard diet (Purina chow, St. Louis, MO, USA)
water: ad libitum
light/dark cycle: 12h light/12 hours dark
Route of induction exposure:
dermal
Remarks:
back
Route of challenge exposure:
inhalation
Remarks:
challenge with Metacholine (Mch)
Vehicle:
other: IPDI: Acetone:Olive oil(4:1, v/v); Metacholine: air/saline
Concentration:
Experiment 1 (preliminary study): IPDI not tested
Experiment 2: IPDI (2%)
Experiment 3: IPDI (1%, 2%)
No. of animals per dose:
Experiment 2 and 3: 6 mice per group and per dose
Details on study design:
Experiment 2:
- Day 0 and day 5: 100µl IPDI applied to shaved back
- Days 10, 11, 12: 12.5µl IPDI applied to each side of both ears
- Day 19: 100µl IPDI applied to shaved back
- Days 24, 25, 26: 12.5µl IPDI applied to each side of both ears
- Day 28: mice assessed for Metacholine (Mch) responsiveness, bronchoalveolar lavage fluid (BALF) and serum collected
- BALF assessed for total and differential cell counts
- total serum antibody IgE and IgG determined
- cytokine message in auricular lymph nodes (IL-4, IL-10, IL-13 and IFN-gamma) determined
Experiment3:
- Day 0 and day 5: 100µl IPDI applied to shaved back
- Days 10, 11, 12: 12.5µl IPDI applied to each side of both ears
- Day 14: auricular lymph nodes removed, cells cultured and assessed for production of cytokine proteins (IL-4, IL-10, IL-13 and IFN-gamma)

In vivo airway responsiveness:
-responsiveness to increasing concentrations (10-100 mg/ml) of aerosolized Methacholine (Mch) was assessed in unanesthetized unrestrained mice in a 12-chamber whole body plethysmograph system (Buxco Electronics, Sharon, CT) 48 h after diisocyanate exposure on day 28.

Serum collection and bronchoalveolar lavage::
- mice anesthetized by i.p. injection of 0.5 ml sodium pentobarbital (5 mg/ml)
- blood sampling by cardiac puncture
- samples placed in serum separator and kept on room temperature for 1-2 h prior to centrifugation
- serum collected and stored at -70°C
- Bronchoalveolar lavage: modified blunted 24 gauge needle was inserted into the trachea and tied in place with surgical silk; lungs were lavaged twice with a single aliquot of 1 ml HBSS, samples were stored on ice until centrifuged at 100xg for 15 min at 4°C; supernatant was removed and stored at -70°C; cell pellet was resuspended in 1 ml HBSS and total cell counts were obtained using Coulter Counter. Air dried stained slides were made from the cells. Differetial cell counts were performed

Total IgE and IgG assays, assessment of cytokine mRNA message and Detection of cytokine proteins: performed as described in the materials and methods chapter of Selgrade et al., 2006

statistics: data analyzed using analysis of variance (ANOVA); Kruskal-Wallis test was used if needed; level of significance for evaluation of factors or paiwise comparisons was set at p<0.05
Challenge controls:
not reported / not applicable
Positive control substance(s):
other: not applicable; no positive controls used
Negative control substance(s):
2,4-dinitrochlorobenzene (DNCB)
other: vehicle
Results:
Experiment 2 (28 day protocol):
-according to cytokine mRNA expression: High responders (TDI, MDI, HMDI) and low responders (IPDI, TMI, TMXDI)
- IgE responses: HMDI, IPDI, TMI > TDI, MDI, TMXDI
- IgG1 responses: HMDI > IPDI, TMXDI, DNCB > TDI, MDI, TMI
- Airway responsiveness: TMI, HMDI, TMXDI > MDI, TDI, IPDI, DNCB
- based on serum antibody responses, the treatment groups did not cluster in the same groups seen with cytokine mRNA expression
- as with serum antibodies, treatment groups did not cluster (based on Mch responsiveness) in the same manner seen with cytokine m RNA expression

Experiment 3 ( cytokine protein profiles after 14 days):
-treatment groups cluster in a manner similar to Experiment 2 and as described in a previous study (Plitnick et al. 2005)
- cytokine responses for animals treated with 1% MDI, HMDI and IPDI were not significantly different from the responses for animals treated with 2% of the same substance
- INF-gamma was not detected in cultures of DNCB treated mice but was detected in cultures from mice treated with 1% and 2% MDI, which was not expected
- IL-2, IL-12: elevated to similar degree in all cultures
- IL-5: was absent in all (except TDI and MDI)
-MCP -1: undetectable in all cultures
- IFN-gamma response detected after culture of cell for 120h did not differ among the different exposure groups
- after culture of cells for 120 h: IL-4, IL-5, IL-10 . IL-13 not detectable; IL-2 elevated to a similar degree in all cultures; MCP-1 detectable

LLNA:
- Stimulation index (SI): 1% TDI (25); 2% MDI (45); 2% HMDI (38); 2% IPDI (50); 1% TMI (23); 1% TMXDI (28); 1% DNCB (32)
Positive control results:
not applicable
Negative control results:
IgE responses: significantly greater in all treated groups than in vehicle and DNCB groups; vehicle and DNCB groups not significantly different from each other
IgG1 responses: IgG1 titers for all treatment groups were different from vehicle
Airway (Mch) responsiveness: groups treated with MDI, TDI, IPDI or DNCB were not statistically more responsive to Mch than vehicle treated group

Responses obtained from Experiment 2 (28 day protocol)

Th2 profile:                     TDI (+); MDI (+); HMDI (+); IPDI (-); TMI (-); TMXDI (-); DNCB (+)

IgE:                     TDI (++); MDI (++); HMDI (+++); IPDI (+++); TMI (+++); TMXDI (++); DNCB (+)

Hyperresponsiveness:     TDI (+); MDI (+); HMDI (++); IPDI (+); TMI (++); TMXDI (++); DNCB (+)

Note: + describes magnitude of significant response; + indicates trend but not significant

Conclusions:
All of the isocyanates tested here (TDI, MDI, HMDI, IPDI, TMI, TMXDI) appear to have some potential to induce respiratory hypersensitivity based on the endpoints measured in these experiments. All induced an increase in total serum IgE relative to DNCB. Using the dermal route of exposure to screen chemicals for the capacity to cause respiratory hypersensitivity has promise. However, additional work is needed to optimize the exposure protocol and select the most appropriate endpoints to assess. Hyperresponsiveness to metacholine (Mch) has some appeal because it assesses a respiratory endpoint and can be done in the same animal used to assess immune endpoints.
Executive summary:

Cytokine profiling of local lymph node responses has been proposed as a simple test to identify chemicals, such as low molecular weight diisocyanates, that pose a significant risk of occupational asthma. The present study was conducted to test the hypothesis that relative differences in the cytokine profile are predictive of relative differences in total serum immunoglobulin(Ig)E and respiratory responses to methacholine (Mch) following dermal exposure to the chemicals.

The data of this study support the findings of Plitnick et al. (2005), which reported TDI, MDI, HMDI; IPDI; TMI; TMXDI can be divided into high and low responders based on Th2 cytokine profiles. However the relative differences in the Th2 cytokine profiles were not predictive of relative differences in either total serum IgE or respiratory responses to Mch following dermal exposure to the chemicals. In addition total serum IgE itself was not an accurate predictor of Mch hyperresponsiveness. These results suggest that the use of cytokine profiling for hazard identification may be premature and that other approaches to hazard identification should be pursued.

The data of this study show that respiratory hyperresponsiveness can occur as a result of dermal exposure to certain isocyanates. Under the dermal exposure conditions reported in this study HMDI, TMI and TMXDI demonstrated an overall greater potential for respiratory effects than TDI, MDI and IPDI. But neither MDI nor TDI, bothof which are known asthmagens, produced a statistically significant increase in hyperresponsiveness to Mch, when applied dermally. Hence, the present dermal exposure regimen is not sufficient (using Mch hyperresonsiveness as an endpoint) to identify all chemicals which might pose a risk via respiratory route.

Regardless of the end points chosen for screening chemicals, more work is needed to identify appropriate positive and negative controls and criteria for determining whether an unknown is more like the negative or more like the positive control.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Partly cited from SIAR for SIAM 23 (Jeju, Korea, October 17-20, 2006):


 


Studies in Animals


 


"Respiratory tract sensitization of guinea pigs following intradermal induction (1%, 100 µl) was studied by Bayer AG (1996) in accordance with the exposure criteria defined in OECD TG 403. High titer IgG1 antibody observed proved that successful sensitization had occurred. However, when challenged by nose only inhalation of aerosol at varying concentrations, the incidence of immediate-onset respiratory reactions was roughly the same in all groups. No delayed-onset reactions, deaths or anaphylactic reactions were observed. Challenge with acetylcholine did not show specific respiratory responses indicating that the animals were hyperrespondive to cholinergic acetylcholine stimuli. Severe reactions were observed with trimellitic anhydride (CAS No. 552-30-7) when investigated with the current animal model, using the equivalent induction and challenge."


The study of Plitnick et al. (2005) is based on the hypothesis on the idea that respiratory sensitizers can be identified based on relativly high expression of cytokines characteristic of Th2 cells. Thus, cytokine profiling may be an effective way to detect respiratory sensitizers. After exposure with the stest item Isophorone Diisocyanate auricular lymph nodes were removed from the animals, the total RNA was extracted and Cytokine mRNA was analysed. Response after exposure with the test item Isophorone Diisocyanate was variable but was comparatively weaker as for strong respiratory sensitizers used in this study. Biological relevance of this results are still under discussion but in general as the result of this study the authors conclude that the test item Isophorone Diisocyanate has at least respiratory sensitizing potential under the conditions of the study.


De Jong et al. (2009) and Arts et al. (2008) reported an induction of cytokine IL-4 after inhalation exposure of IPDI in an inhalative LLNA study, which is discussed to be an indicator of respiratory sensitizers.


Further studies examined cytokine profiles, serum antibodies and respirative responses after dermal exposure with IPDI followed by intranasal challenge with IPDI (Farraj et al. 2007) or followed by inhalation of metacholine (Selgrade et al. 2006) in order to predict and screen for a respiratory sensitizing potential by using the dermal route of exposure. Although the results were ambigous and further experimental work is needed the authors conclude that IPDI appears to have some potential to induce respiratory hypersensitivity.


 


Studies in Humans


 


"A 50-year old spray painter developed severe asthma soon after introduction of a new paint containing 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate. His asthma was associated with an abnormal chest X-ray, blood eosinophilia, normal IgE level, negative skin prick tests and no precipitins to Aspergillus fumigatus. After successful initial therapy, the person was left in an enclosed room for 30 minutes each on three days, followed by spirometry at hourly intervals for nine hours. Exposure conditions in the enclosed room were as follows:


Day 1: Sitting


Day 2: Painting a chair without 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate in the spraying enamel


Day 3: Painting a chair with 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate in the spraying enamel


Exposure was not quantified. On day 3, the patient required treatment 3 hours 35 minutes after cessation of challenge. A very large reduction in forced expiratory volume was observed on that day (Clarke and Aldons, 1981).


Germanaud et al. (2003) published a case of occupational hypersensitivity pneumoapathy, which according to the investigators is rarely caused by isocyanates. A 50 year old man had worked in the production of polyurethane foams and polyurethane coatings for 32 years with a generally low exposure. He then was engaged more closely in a polyurethane synthesis from 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate. Few hours after the beginning of this new occupational exposure, which was not defined any more specifically, he showed dyspnea, fever (39°C), and crepitant rales. Further investigations revealed ground glass appearance on the thoracic CT scan and lymphocytosis in the broncho-alveolar lavage. Effects were confirmed by transbronchial biopsy. Only the functional assessment (airflow obstruction and absence of marked reduction in CO transfer) was atypical for hypersensitivity pneumapathies.


A poorly documented case is also reported by Tyrer (1979): In 1974, a sprayer in a firm of motor body repairers used for some months intermittently a two-pack paint containing 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (not quantified), toluene and xylene, with no ill-effects. The spraying was done in a large, completely enclosed booth with effective downdraught through the floor. He then developed tightness of the chest and dyspnea, which disappeared when he took a few days off, but recurred, shortly after his return to work. The sprayer who took his place had similar symptoms in a milder form, which lasted only a few hours. A causal relationship between the asthmatic symptoms and a specific substance was not established in this mixed exposure case."



Migrated from Short description of key information:
Cited from SIAR for SIAM 23 (Jeju, Korea, October 17-20, 2006):
"No validated animal model is available to assess the potential for respiratory sensitization or asthma in humans, and one animal study did not show respiratory tract sensitization when exposed by inhalation (challenge) following intradermal induction. However, due to the well known reactivity of diisocyanates, respiratory sensitization is likely to occur."

Justification for classification or non-classification

The substance 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate is classified according to the criteria of Regulation (EC) No 1272/2008 (CLP) as follows: Resp. Sens. 1; H334 and Skin Sens. 1; H317

Categorisation of IPDI concerning skin sensitization

Data:

Animal data

CLP Sub-category

LLNA, EC3 > 0.1 and < 0.25 %

1A

GPMT, intradermal induction: 10 %, sensitization rate: 80 %

1B

BT (Buehler test), induction conc.: 5 %, sensitization rate: 80 %

1A

 

 

Human data

CLP Sub-category

Schlede et al. (2003): proven contact allergenic effect less frequently, cross-reactivity

1B

 

Discussion:

The results of GPMT and BT are contradictory. Both assays have been developed for the assessment of sensitization potential (yes or no) and not for potency determination. Even though there are criteria defined in Annex I to Regulation (EC) No 1272/2008 (CLP) these values cannot have been considered in the study design and are thus less appropriate to distinguish between categories 1A and 1B

 

The test system LLNA reveals positive results for skin as well as respiratory sensitizers and it cannot be conclusively evaluated whether the indicated potency directly relates to skin sensitization. Moreover, it is questionable, whether the LLNA is over-predictive for irritants.

Additionally, ICCVAM concluded in its report of 2011 (NIH Publication No. 10-7512

http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-app/TMER.htm) that the “LLNA cannot be considered a stand-alone assay to determine skin sensitization potency categories. … Among the 21 substances that produced a LLNA EC3 ≤ 2 %, 67 % (14/21) were correctly identified as strong sensitizers, but 33 % (7/21) were incorrectly overclassified as strong skin sensitizers based on available human test data.”

The result of the LLNA (pre-guideline study with minor deviations from OECD 429 guideline) of IPDI, a known respiratory sensitizer with corrosive properties, point to a high potency (correlate to sub-category 1A).

Schlede et al. (2003) evaluated IPDI mainly based on the available human data to be a proven human sensitizer not with high but moderate frequency in humans. This would correlate to sub-category 1B.

Overall as the data on potency of IPDI are limited and human data and animal data are not fully consistent it is concluded that the available data currently do not allow a solid assessment of the potency.

According to Annex I to Regulation (EC) No 1272/2008 (CLP) paragraph 3.4.2.2.1.1“skin sensitisers shall be classified in Category 1 where data are not sufficient for sub-categorisation.”.

Therefore, a classification of IPDI in Category 1 of hazard class “skin sensitization” is appropriate here.