Registration Dossier

Administrative data

Description of key information

In a guinea pig maximisation test according to OECD TG 406, sensitisation was observed in 18 of 20 animals 24 hrs after using a challenge concentration of 5 %. With a challenge concentration of 2.5 %, 7 of 20 animals were positive (Hüls AG, 1983). The sensitising properties of 3-aminomethyl-3,5,5-trimethylcyclohexylamine were also observed in two other guinea pig maximisation tests (Inveresk, 1981; Thorgeirsson, 1978) as well as in a reduced form of LLNA according to OECD 429, in which the test item revealed sensitizing properties in concentrations of 10% (CiToxLab, 2016).

From a number of publications there is evidence that frequent occupational exposure to 3-aminomethyl-3,5,5-trimethylcyclohexylamine may lead to the development of allergic contact dermatitis in humans.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-09-19 - 1983-10-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no positive control group (not required by 1981 version of guideline)
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
yes
Remarks:
no positive control group (not required by 1981 version of guideline)
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 1981 and is sufficient for evaluation of the skin sensitisation potential of the test substance.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS: 
- Strain: Dunkin-Hartley (Bor: DHPW)
- Source: Winkelmann, Borchen (Germany)
- Weight at study initiation: 363 g (mean)
- Diet: ad libitum, special diet for guinea pigs, SSniff G 4 (Ssniff, Soest, Germany)
- Water: ad libitum, tab water
- Acclimation period: 5-8 days
ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 1 °C
- Humidity: 60 +/- 5 %
- Photoperiod: 12 hours artificial light, 12 hours dark
Route:
other: intracutaneous (1st) and occlusive epicutaneous (2nd)
Vehicle:
other: 10 % ethanol
Concentration / amount:
1st application: Induction 0.1 % intracutaneous
2nd application: Induction 7.5 % occlusive epicutaneous
3rd application: Challenge occlusive epicutaneous
Route:
epicutaneous, occlusive
Vehicle:
other: 10 % ethanol
Concentration / amount:
1st application: Induction 0.1 % intracutaneous
2nd application: Induction 7.5 % occlusive epicutaneous
3rd application: Challenge occlusive epicutaneous
No. of animals per dose:
9 (10) control group, 1 animal died after 1 week, cause of death is unkwon
20 test group
Details on study design:
- Induction schedule: injection followed 1 week later by patch treatment (0.3 ml) for 48 hours;
- Injection details: 0.1 ml each at 6 positions on shoulders:
2 x Freund's Complete Adjuvant
2 x test substance in 10 % ethanol
2 x Freund's Complete Adjuvant / 0.2 % test substance (1:1)
simultaneous and symmetrical application of each solution
controls: 10 % ethanol instead of test substance
- Challenge schedule: 2 weeks after end of induction patch treatment for 24 hours
- Concentrations used for challenge: 2.5 and 5 %; readings 24, 48, and 72 hours after removal of patch
- Rechallenge: no
EXAMINATIONS
- Grading system: possible scores 0 / 1 / 2 / 3
- Pilot study: dose range finding study
Challenge controls:
9 (10) animals, 1 animal died after 1 week, cause of death is unkwon
vehicle treatment
Positive control substance(s):
no
Positive control results:
not applicable
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2.5 %
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2.5 %. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2.5 %
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2.5 %. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
2.5%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 2.5%. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2.5% test substance used for challenge
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 2.5% test substance used for challenge. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2.5% test substance used for challenge
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 2.5% test substance used for challenge. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
2.5% test substance used for challenge
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: see Remark
Remarks:
Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 2.5% test substance used for challenge. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 %
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5 %. No with. + reactions: 18.0. Total no. in groups: 20.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5 %
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 %. No with. + reactions: 15.0. Total no. in groups: 20.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
5 %
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5 %. No with. + reactions: 10.0. Total no. in groups: 20.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5% test substance used for challenge
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 5% test substance used for challenge. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5% test substance used for challenge
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5% test substance used for challenge. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: see section "Remarks on results including tables and figures".
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
5% test substance used for challenge
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
see section "Remarks on results including tables and figures"
Remarks on result:
other: see Remark
Remarks:
Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 5% test substance used for challenge. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: see section "Remarks on results including tables and figures".


    
    
    

Local effects:
- 24 hours after intracutaneous applications, animals mainly from the test substance treated group displayed poorly healing necrotic inflammations.

- After the induction patch treatment for 48 hours, the animals treated with the test substance had bleeding and matter discharging inflammations at the places of injections leading to thick crusts.

- In general after removal of the patch, severe inflammation and itching were observed, causing particularly the animals treated with test substance to scratch their skins open in the area of injection.

- after challenge scale formation and desiccation of skin was observed during the observation period on some animals

Conclusions:
Isophorone diamine has to be considered to be sensitizing to the skin of guinea pigs under the conditions of the study.
Executive summary:

The skin sensitizing properties of isophorone diamine were determined in a guinea pig maximation test according to OECD TG 406 (positive controls not required by 1981 guideline version). Twenty male guinea pigs were intradermally injected with isophorone diamine at 0.1 % in 10% ethanol and one week later epidermally exposed to a 7.5 % concentration of test substance for 48 hours (occlusive). Ten control animals were similary treated, but with vehicle alone. Two weeks after the epidermal application all animals were challegend with 2.5 % and 5 % test substance and with vehicle (24 hours occlusive). At challenge concentration of 2.5 % 7/20 animals showed a sensitization 24 hours after the patch test, 5/20 animals 48 hours after the test and 2/20 72 hours after the test. At challenge concentration 5 % 18/20 animals showed a sensitization 24 hours after the patch test, 15/20 animals 48 hours after the patch test and still 10/20 72 hours after the test. No animal of the control group showed any positive reaction. Therefore, Isophorone diamine has to be considered to be sensitizing to the skin of guinea pigs under the conditions of the study.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981-01-14 - 1981-02-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no positive control group (not required by 1981 version of guideline)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
yes
Remarks:
no positive control group (not required by 1981 version of guideline)
GLP compliance:
no
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
- Source: Porcellus Animals Limited (UK)
- Weight at study initiation: 300-350 g
- Dose group: 20 animals
- Controls: 10 animals, Freund's Complete Adjuvant
- Diet: ad libitum, special diet for guinea pigs, BP Nutrition FD1, supplemented with hay
- Water: ad libitum, tab water
- Acclimation period: 5-8 days
ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C (17.5 - 22°C)
- Humidity: 50 % (40% - 59 % )
Route:
other: 1st intracutaneous, 2nd occlusive epicutaneous
Vehicle:
water
Concentration / amount:
1st application: Induction 1 % intracutaneous
2nd application: Induction 1 % occlusive epicutaneous
3rd application: Challenge 5% and 10 % occlusive epicutaneous
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
1st application: Induction 1 % intracutaneous
2nd application: Induction 1 % occlusive epicutaneous
3rd application: Challenge 5% and 10 % occlusive epicutaneous
No. of animals per dose:
20 test group
10 control group
Details on study design:
- Induction schedule: intradermal injection followed after one week by occlusive patch treatment for a further 48 hours
- Injection details: 0.1 ml each at 6 positions in scapular region:
2 x Freund's Complete Adjuvant (FCA)
2 x test material (1% in dist. water)
2 x 1:1 emulsion of FCA / test material (1% in dist. water)
simultaneous and symmetrical application of each solution;
control and dose finding animals: 2 x 0.1 ml FCA only each
- Challenge schedule: 3 weeks after injection occlusive patch treatment for 24 hours, reading 24 hours after removal of patch
- Concentrations used for challenge: 10% and 5%
- Rechallenge: no
Challenge controls:
Vehicle treatment
Positive control substance(s):
no
Positive control results:
not applicable
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
See section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5 % . No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: See section "Remarks on results including tables and figures".
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
See section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 5 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: See section "Remarks on results including tables and figures".
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10 %
No. with + reactions:
12
Total no. in group:
20
Clinical observations:
See section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 12.0. Total no. in groups: 20.0. Clinical observations: See section "Remarks on results including tables and figures".
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
See section "Remarks on results including tables and figures"
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: See section "Remarks on results including tables and figures".
RESULTS OF PILOT STUDY: no responses regarding skin irritation were observed at any concentration used in the pre-test (10%, 5%, 2%, 1% test substance)
RESULTS OF TEST
- Sensitization reaction:
-12/20 animals positive (i.e. erythema) at 10% challenge concentration
-no animals positive at 5% challenge concentration
-no erythema in control group
Conclusions:
It is concluded that the results of the study are indicative of skin sensitisation and that Isophorone diamine is considered to be a dermal sensitiser in guinea pigs under the conditions of the study.
Executive summary:

The skin sensitizing properties of isophorone diamine were determined in a guinea pig maximation test according to OECD TG 406 (positive controls not required by 1981 guideline version). Twenty female guinea pigs were intradermally injected with isophorone diamine at 1 % in dist. water and one week later epidermally exposed to a 1 % concentration of test substance for 48 hours (occlusive). Ten control animals were similary treated, but with vehicle alone. Two weeks after the epidermal application all animals were challegend with 5 % and 10 % test substance and with vehicle (24 hours occlusive). No control group animal showed erythema at either 10 or 5 % challenge concentration, no erythema was noted in test group animals after challenge with 5 % test item, in test group challenged with 10 % isophorone diamine 12/20 animals showed erythema. Therefore, isophorone diamine is considered to be a dermal sensitizer in guinea pigs under the conditions of the study.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
approx. 1977 / 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
According to Magnusson B, Kligman AM (1969). J. Invest. Dermatol. 52, 268.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
not specified
Sex:
not specified
Route:
other: 1st intracutaneous, 2nd epicutaneous, occlusion not reported
Vehicle:
other: acetone, CAS No. 67-64-1
Concentration / amount:
1st application: Induction 0.5 % intracutaneous
2nd application: Induction 0.5 % other: epicutaneous, occlusion not reported
3rd application: Challenge 2 % occlusive epicutaneous
Route:
epicutaneous, occlusive
Vehicle:
other: acetone, CAS No. 67-64-1
Concentration / amount:
1st application: Induction 0.5 % intracutaneous
2nd application: Induction 0.5 % other: epicutaneous, occlusion not reported
3rd application: Challenge 2 % occlusive epicutaneous
No. of animals per dose:
no information
Details on study design:
TEST ANIMALS:
- Controls: vehicle and Freunds Complete Adjuvant
ADMINISTRATION/EXPOSURE
- Induction schedule: not reported
- Challenge schedule: two weeks after the second stage of sensitization, 24-hour patch test, evaluation 24 hours after removal of patch
EXAMINATIONS
- Grading system: obvious redness and swelling judged by two persons independently
Challenge controls:
described
Positive control substance(s):
no
Positive control results:
not applicable
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.5 %
Remarks on result:
positive indication of skin sensitisation
RESULTS OF TEST
- Sensitization reaction: 100 % of the animals positive
Conclusions:
Isophorone diamine is considered to be a dermal sensitizer for guinea pigs under the conditions of the study.
Executive summary:

The skin sensitizing properties of isophorone diamine were determined in a guinea pig maximation test. Guinea pigs were intradermally injected with isophorone diamine 0.5 % in acetone and later epidermally exposed to a 0.5 % concentration of test substance occlusive. Control animals were similary treated, but with vehicle alone regarding to the induction. Two weeks after the epidermal application all animals were challegend with 2 % test substance (24 hours occlusive). All test animals showed positive reactions. Therefore, isophorone diamine is considered to be a dermal sensitizer for guinea pigs under the conditions of the study.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2016-03-23 to 2016-04-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
yes
Remarks:
A reduced form of LLNA (using two milestone concentrations) was performed . Study was a ranking test. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formerly: Harlan Laboratories S.r.l.)
- Females nulliparous and non-pregnant: yes

- Age at study initiation: 9 weeks
- Weight at study initiation: 18.0 - 20.9 g
- Housing: Group caging/ mice were provided with glass tunnel-tubes
- Diet: ad libitum (ssniff SM Rat/Mouse- Breeding & Maintenance complete diet)
- Water: ad libitum (tap water from municipal supply)
- Acclimation period: 14 days
- Indication of any skin lesions: Only healthy animals were used for the study. Health status was certified by the veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 - 24.8
- Humidity (%): 23 - 76
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark cycle
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2 and 10% of test item in olive oil
No. of animals per dose:
4 female mice
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
Based on the available information and as agreed by the Sponsor, Acetone: Olive oil 4:1 (v/v) mixture (abbreviated as AOO) was selected for vehicle of the study. Two milestone concentrations (10 and 2% (w/v)) were selected by the Sponsor for the study.

- Two groups received the test item formulated in AOO at 10 and 2% (w/v) concentrations,
- the negative control groups received the vehicle (AOO),
- the positive control group received 25% (w/v) alpha- Hexylcinnamaldehyde solution (HCA) (dissolved in AOO).
The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

Formulation
To prepare the dosing formulations, the test items were freshly diluted with the selected vehicle to obtain appropriate concentrations in the Pharmacy of CiToxLAB Hungary Ltd. They were made shortly before application to mice (within 1 hour) and were considered to be stable for this short period. The applicable dose levels (two threshold concentrations) were selected by the Sponsor in accordance with the aim of the ranking study.
The test items were weighed and formulations prepared daily on a weight:volume basis as % (w/v) in the Pharmacy of CiToxLAB Hungary Ltd.
Analytical determination of concentration, stability and homogeneity of the individual test item formulations was not performed because of the character and the short period of study, although the formulations were checked for visible homogeneity and physical stability. Test item formulations at 10 and 2% (w/v) concentrations appeared by visual inspection to be clear colourless solutions.
The negative control group received only the vehicle (Acetone: Olive oil 4:1 (v/v) concurrent to the test item treatment group.
Animals assigned to the positive control group were treated with 25% (w/v) α-Hexylcinnamaldehyde solution (dissolved in AOO) concurrent to the test items treated groups. The relevant data of the positive control substance are listed below:
Name: α-Hexylcinnamaldehyde
Synonym: α-Hexyl cinnamic aldehyde
Abbreviation: HCA
CAS No.: 101-86-0
Batch No.: MKBS3936V
Manufacturer: Sigma-Aldrich Co.
Nominal purity: ≥95%
Purity: 97.3%
Expiry: 31 July 2017

Clinical observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed at least twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
Both ears of each mouse were observed for erythema and scored using the following table:

Erythema Scoring
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar 4
formation preventing grading of erythema

Note: Excessive local skin irritation is indicated by an erythema score ≥ 3 on any day of measurement

Proliferation Assay:
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.
Ear thickness was measured in the study using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was also performed by ear punch weight determination on Day 6 after the euthanasia of the experimental animals.
Excessive local skin irritation is indicated by an increase in ear thickness of ≥ 25% on any day of measurement.
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes of mice were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed.
Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The proliferative response of lymph node cells from the lymph nodes of each individual animal was expressed as radioactive disintegrations per minute (DPM) per animal. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as disintegrations per node (DPN = DPM divided by the number of lymph nodes) for each animal, following the industry standard for data presentation.
Stimulation index (SI = mean DPN of treated group divided by mean DPN of the appropriate control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
The use of the individual approach to calculate SI made the use of a statistical analysis available. The statistical analysis was performed using the SPSS/PC+ (4.0) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test.
Key result
Parameter:
SI
Value:
7.9
Test group / Remarks:
in 10% test group
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
in 2% test group
Parameter:
other: Group DPN
Value:
8 318.8
Test group / Remarks:
in 10 % test group
Parameter:
other: Group DPN
Value:
1 399.3
Test group / Remarks:
in 2 % test group
Key result
Parameter:
EC3
Value:
4.1

Clinical observations

No mortality or signs of systemic toxicity were observed during the study.

Body weight meassurements

No clear treatment related effects on body weight were observed in the study. Only one animal in the 10% (w/v) VESTAMIN IPD group showed marked body weight loss (≥5%), but this was considered as animal variability.

EAR THICKNESS MEASUREMENTS

Ear thickness of the animals was measured using by a thickness gauge and by ear punch weight determination. Significantly increased ear thickness values (increase of ≥25%) were recorded for for all animals in the 10% (w/v) Test Item D (VESTAMIN IPD) dose group on Day 6.

Erythema (maximum score of 1), was recorded for Test Item D (VESTAMIN IPD) for all animals in the 10% (w/v) dose group on Days 4-5 and for one animal in the 2% (w/v) dose group on Day 4.

Conclusions:
In conclusion the test item revealed sensitizing properties in the local lymph node assay in concentrations of 10% (showing a Si of 7.9)
Executive summary:

The purpose of the study was to determine the ranking of five substances according to their sensitizing potency. Therefore, only a reduced form of LLNA was performed, which is sufficient for the purpose and reduced the number of animals used in the study.

The study was performed according to the OECD guideline 429.

Based on the available information and as agreed by the Sponsor, Acetone: Olive oil 4:1 (v/v) mixture (abbreviated as AOO) was selected for vehicle of the study. Two milestone concentrations (10 and 2% (w/v)) were selected by the Sponsor for the study.

In the performed experiment female CBA/CaOlaHsd mice were used.

- two groups received the individual test item (Test Items  formulated in AOO at 10 and 2% (w/v) concentrations,

- the negative control groups received the vehicle (AOO),

- the positive control group received 25% (w/v) HCA (dissolved in AOO).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. No treatment related body weight loss was observed in the test item treated animals.

Minor wound was observed on all animals in the 10% (w/v) dose group on Days 4-6, slightly fixed ears were also recorded on Days 5-6. There was no indication of any irritancy at the site of application by visual examination, but ear thickness measurements indicated some local irritation for all animals in the 10% (w/v) dose group on Day 6.

The stimulation index (SI) values measured at 10 and 2% concentrations were 7.9 and 1.3 for the Test Item.

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

Studies in humans

Cited from SIAR for SIAM 18 (Paris, April 2004):

"There are several publications describing 3-aminomethyl-3,5,5-trimethylcyclohexylamine related contact dermatitis: Three out of 15 workers manufacturing plastic tennis rackets developed allergic contact dermatitis when exposed concomitantly to 3-aminomethyl-3,5,5-trimethylcyclohexylamine and epoxy resin. Symptoms appeared 3 months, 6 weeks or 3 weeks after starting this work and disappeared completely within 3 weeks after moving to a different department. Patch tests were positive with 1, 2, or 5 % 3-aminomethyl-3,5,5-trimethylcyclohexylamine in both ethanol and olive oil. After the test it became obvious that the controls were also sensitised (Lachapelle, Tennstedt and Dumont- Fruytier, 1978). Two of the three workers positive for 3-aminomethyl-3,5,5-trimethylcyclohexylamine from the Lachapelle, Tennstedt and Dumont-Fruytier study (1978) and two additional ones who were also positive for 3-aminomethyl-3,5,5-trimethylcyclohexylamine, were patch tested 1 month later with isophorone diisocyanate (1 %). All four were positive, while five control volunteers were negative. This study may indicate a cross-sensitivity between 3-aminomethyl-3,5,5-trimethylcyclohexylamine and the corresponding diisocyanate, though its documentation is poor (Lachapelle and Lachapelle- Ketelaer, 1979). From 142 persons considered to have skin disorders from current occupational exposure to epoxy compounds, 135 had allergic contact dermatitis. 53 of those were patch tested for 3-aminomethyl- 3,5,5-trimethylcyclohexylamine, and three were positive towards this substance. Patch tests with 0.5 % 3-aminomethyl-3,5,5-trimethylcyclohexylamine were used, as this concentration was observed to induce neither irritation nor active sensitisation in the patch testing (Jolanki, 1991). A 38-year old bricklayer had prolonged skin contact with a work shoe contaminated with a 3- aminomethyl-3,5,5-trimethylcyclohexylamine containing two-component glue. The man developed a dermatitis on the chest, upper back, arms and legs and was tested positive for 3-aminomethyl- 3,5,5-trimethylcyclohexylamine (0.5%) 2 months after the generalised skin reaction had healed (Kelterer, Bauer and Elsner, 2000; short communication). A total of 137 employees of 10 companies preparing and using epoxy resins for coating, flooring, impregnating and repairing concrete, brick and wooden structures were examined. Positive patch tests were observed in 27 of the 137 employees, predominantly with epoxy resin (25 persons / 18.5 %), but also with 0.1 % 3-aminomethyl-3,5,5-trimethylcyclohexylamine (3 persons / 2.3 %). Positive correlation with the development of an allergy were observed with the frequency of exposure, the duration of employment and the permanent wearing of gloves (van Putten, Coenraads and Nater, 1984). 15 A 53-year-old male employed for laying impermeable floors had a very itchy, symmetrical, erythematous, edematous eruption of the face, which had begun during work. Repeatedly, it had healed within few weeks off work but recurred when returning to work. No working colleagues had similar problems, though in the past, a man had left the job following a similar history. From several patch tests with standard series and with his working materials, only 3-aminomethyl-3,5,5- trimethylcyclohexylamine was positive with erythema from 0.1 %, and erythema, oedema and vesicles from 1 % and 5 % after three days (Patussi et al., 1995). A 44-year old man working at a lamination machine, having had no previous skin problems except photosensitisation, after 2 years at the lamination machine observed eczema on the hands and wrists, as well as on the right upper arm. Enclosure of the process was improved. Four years later, his dermatitis worsened covering particularly typical sites of sun exposure. It healed during 2 weeks off work. In patch testing, positive reactions were observed for 3-aminomethyl-3,5,5- trimethylcyclohexylamine, the two glue components, plastic-laminated fiber cloth as such, perfume mix, and epoxy resin (Tarvainen et al., 1998). During January 1984 to May 1992, Tosti et al. (1993) patch tested 39 patients with occupational allergic contact dermatitis to epoxy resin system substances. Positive reactions for 3-aminomethyl- 3,5,5-trimethylcyclohexylamine were observed in 0/18 persons from electronics industry, 1/8 painters, 0/3 from fiberglass handling, 1/8 from gluing, 0/1 dental technicians, and 1/1 mechanics. The documentation of this study is not sufficient to allow a judgment on its validity. In a further poorly documented study, all persons from the contact dermatitis database at Waikato Hospital (Hamilton, NZ) with relevant and work related contact dermatitis to epoxy resin compounds, i.e. 16 males, average age 37 years (range 21-53 years), were subject to several patch tests. Among them, one reacted positive with 3-aminomethyl-3,5,5-trimethylcyclohexylamine (0.1 % in petrolatum) (Rademaker, 2000). Kanerva et al. (1999) studied the relative frequency of occupational contact dermatitis towards 53 sensitisers by patch testing all patients exposed to plastics and remitted to an occupational dermatology clinic during the years 1991 - 1996. 311 patients were tested using occlusive patches with 0.5 % 3-aminomethyl-3,5,5-trimethylcyclohexylamine in petrolatum for 48 hours followed by three readings, usually on days 2, 3, and 4-6. 3-Aminomethyl-3,5,5-trimethylcyclohexylamine was among the 26/53 substances which induced no allergic reaction, but it induced irritation in one case. Maximum frequencies were 5.1 % for sensitisation and 9.5 % for irritation."

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Studies in humans

Cited from SIAR for SIAM 18 (Paris, April 2004):

"A 44-year old man had a serious attack of bronchial obstruction after working with resins and hardeners, releasing fumes of a mixture of trimethyl-1,6-hexanediamine and 3-aminomethyl-3,5,5- trimethylcyclohexylamine. Eight hours after deliberate challenge with the hardener, a large increase of airway resistance was found. Seventy-two hours after challenge, eosinophilia in the bronchoalveolar fluid together with a decrease in peripheral eosinophils was seen. After cessation of contact with this hardener, no more acute episodes were seen, though maintenance treatment with a topical corticosteroid and a beta2-agonist remained necessary (Aleva et al., 1992).


Cited from SIAR for SIAM 18 (Paris, 2004):
"Since there is only one publication on possible airway effects of 3-aminomethyl-3,5,5-trimethylcyclohexylamine (describing a single human case) no definite conclusion can be drawn on respiratory sensitisation."

Justification for classification or non-classification

Because of outcome of the skin sensitisation studies the substance isophorone diamine is classified as Skin Sens. Category 1A, (H317: May cause an allergic skin reaction) according CLP regulation (1272/2008).