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Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Method: Magnusson B, Kligman AM (1969). J. Invest. Dermatol. 52, 268.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Species:
guinea pig
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
no data
Route:
intradermal and epicutaneous
Vehicle:
water
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:
water
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:
15
Details on study design:
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:
similarly treatment, but with vehicle and Freunds Complete Adjuvant alone
Positive control substance(s):
not specified
Positive control results:
not examined
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
2 %
No. with + reactions:
12
Total no. in group:
15
Clinical observations:
80 % of the animals showed a positive sensitization reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2 %. No with. + reactions: 12.0. Total no. in groups: 15.0. Clinical observations: 80 % of the animals showed a positive sensitization reaction.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle and Freunds Complete Adjuvant
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
no reactions
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle and Freunds Complete Adjuvant. No with. + reactions: 0.0. Total no. in groups: 15.0. Clinical observations: no reactions.

RESULTS OF TEST
Sensitization reaction: 80 % of the animals positive

Conclusions:
In a guinea pig maximization test (according to Magnusson B and Kligman AM), sensitization was observed in 80% of all animals at 24 hours. Based on these results, 2,2,4(or 2,4,4)-trimethylhexane-1,6 -diamine should be classified as contact sensitizer.
Executive summary:

The skin sensitizing properties of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine were conducted in a guinea pig maximization test according to Magnusson B, Kligman AM (1969). 15 guinea pigs were intradermally injected with a 0.5 % concentration of 2,2,4(or 2,4,4)-trimethylhexane-1,6 -diamine (in water) and epidermally exposed to a 0.5 % concentration of test substance. Control animals were similary treated, but with vehicle and Freunds Complete Adjuvant alone. Two weeks after the epidermal application all animals were challegend with 2 % test substance and the vehicle.

Under the conditions of the test sensitizing effects were observed in 80 % of all animals at 24 hours. Based on these results, 2,2,4(or 2,4,4)-trimethylhexane-1,6 -diamine should be classified as contact sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-09-17 to 1984-10-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions: No positive control (not required by 1981 version of Test Guideline)
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: 
- Strain: Dunkin-Hartley (Bor: DHPW)
- Sex: female
- Source: Winkelmann, Borchen (Germany)
- Weight at study initiation: 428 g (mean)
- Controls: vehicle treatment
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 1 °C
- Humidity (%): 60 +/- 5 %
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
1st application: Induction 0.1 % intracutaneous
2nd application: Induction 10 % occlusive epicutaneous
3rd application: Challenge 2 % occlusive epicutaneous
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
1st application: Induction 0.1 % intracutaneous
2nd application: Induction 10 % occlusive epicutaneous
3rd application: Challenge 2 % occlusive epicutaneous
No. of animals per dose:
20 females (test) / 20 females (control)
Details on study design:
ADMINISTRATION/EXPOSURE 
- Induction schedule: injection followed 1 week later by patch treatment  for 48 hours
- Injection details: 0.1 ml each at 6 positions on shoulders:   
2 x Freund's Complete Adjuvant   
2 x test substance 0.1 % in aqua dest.   
2 x Freund's Complete Adjuvant / 0.2 % test substance (50:50)   
simultaneous and symmetrical application of each solution   
controls: aqua dest. instead of test substance
- Challenge schedule: 2 weeks after end of induction patch treatment for  24 hours
- Concentrations used for challenge: 2 %; readings 24, 48, and 72 hours  after removal of patch
- Rechallenge: no
- Positive control: no
EXAMINATIONS
- Grading system: possible scores 0 / 1 / 2 / 3       
0 % of animals scored > 0: no sensitization  
1 -  8 % of animals scored > 0: very slight sensitization  
9 - 28 % of animals scored > 0: slight sensitization
29 - 64 % of animals scored > 0: distinct sensitization
65 - 80 % of animals scored > 0: severe sensitization
81 -100 % of animals scored > 0: extreme sensitization
Challenge controls:
similarly treatment, but with vehicle alone
Positive control substance(s):
no
Positive control results:
not examined
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
2 %
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
see "Remarks on results"
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2 %. No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: see "Remarks on results".
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Vehicle
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no reactions
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: no reactions.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
2 %
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
see "Remarks on results"
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2 %. No with. + reactions: 16.0. Total no. in groups: 20.0. Clinical observations: see "Remarks on results".
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Vehicle
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no reactions
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: no reactions.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
2 %
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
see "Remarks on results"
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 2 %. No with. + reactions: 8.0. Total no. in groups: 20.0. Clinical observations: see "Remarks on results".
Reading:
other: 3 rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
Vehicle
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no reactions
Remarks on result:
other: Reading: other: 3 rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: Vehicle. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no reactions.

RESULTS OF TEST
- Sensitization reaction: 
  16/20 animals positive at 24 and 48 hours = severe sensitization
  8/20 animals positive at 72 hours
  no animals positive in control group
- Clinical signs: 
  1st induction, FCA application sites: distinct erythema and edema and slight necrosis
  1st induction, FCA + test substance application sites: severe erythema and edema, necroses and brown / black discoloration
  1st induction, vehicle application sites: slight erythema
  2nd induction, test group: inflammation, black discoloration
  2nd induction, control group: slight inflammation at few injection sites

Conclusions:
In a guinea pig maximization test (according to OECD Guideline 406, positive controls not required by 1981 guideline version), sensitization was observed in 16 of 20 female animals. Based on these results, 2,2,4(or 2,4,4)-trimethylhexane-1,6 -diamine should be classified as contact sensitizer.
Executive summary:

The skin sensitizing properties of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine were conducted in a guinea pig maximization test according to OECD 406 (positive controls not required by 1981 guideline version). Twenty female guinea pigs were intradermally injected with a 0.1 % concentration of 2,2,4(or2,4,4)-trimethylhexane-1,6 -diamine (in water) and one week later epidermally exposed to a 10 % concentration of test substance. Twenty control animals were similary treated, but with vehicle alone. Two weeks after the epidermal application all animals were challegend with 2 % test substance and the vehicle.

Under the conditions of the test sensitizing effects were observed in 16 of the 20 animals at 24 and 48 hours. Based on these results, 2,2,4(or 2,4,4)-trimethylhexane-1,6 -diamine should be classified as contact sensitizer.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2016-02-29 to 2016-03-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study described in this report was conducted in compliance with ECVAM (2014), DB-ALM protocol 155: KeratinoSensTM protocol and OECD guideline 442d
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
Analysis of stability, homogeneity and concentration of the test substance under test conditions was not performed as part of this study
GLP compliance:
not specified
Type of study:
activation of keratinocytes
Details on the study design:
BASIS OF THE METHOD
The only feature all skin sensitizers have in common is their intrinsic electrophilicity or their potential
to be metabolically transformed to electrophilic chemicals. The signalling pathway with the repressor
protein Keap1 (Kelch-like ECH-associated protein 1) and the transcription factor Nrf2 (nuclear factor
(erythroid-derived 2)-like 2), which binds to the antioxidant / electrophile response element (ARE /
EpRE), is known to respond to electrophilic chemicals and it was found to be a valuable cellular
Givaudan in vitro toxicology laboratory Study Number: RCR 153’561
KeratinoSensTM test report Page 8 of 32
endpoint to detect skin sensitizers in vitro [8]. This result was confirmed by independent laboratories
[9-13].
EXPERIMENTAL DESCRIPTION
Test System(s):
The KeratinoSens cell line was originally derived from the human keratinocyte culture HaCaT. It
contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene
AKR1C2 [2].
Endpoint & Endpoint Detection:
Two endpoints are measured: (i) Luciferase induction after a 48 h treatment with test chemicals and
(ii) cytotoxicity as determined with the MTT assay recorded in a parallel plate with the same cell
batch and made up with the same dilutions of the test chemicals.
Endpoint Value:
For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration
needed to reach an 1.5 fold induction (EC1.5) are calculated. For cytotoxicity the IC50 value is
extrapolated.

Basic Procedure:
Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a
final level of 1% of the solvent DMSO containing the test chemical. Each compound is tested at 12
concentrations in the range from 0.98 to 2000 μM. Each test plate contains six wells with the solvent
control, 1 well with no cells for background value and 5 wells with a dose response of the positive
control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same
set-up, and a forth parallel plate is prepared for cytotoxicity determination.

Positive control
In each test Cinnamic aldehyde is included as positive control. It is tested in each test plate at five
concentrations from 4 – 64 μM.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the
SOP. The test plates are read by a plate reader, and the generated raw data are directly pasted into
this template, and all data processing is performed automatically by this Excel sheet.
For both the MTT and the luciferase data, first the background value recorded in an empty well
without added cells is subtracted.
For the MTT data the % viability is then calculated for each well in the test plate in relation to
average of the six solvent control wells.
For the luciferase data the average value of the six solvent control wells is set to 1, and for each well
in the test plate the fold induction is calculated in relation to this value.
The following parameters are then calculated from these processed raw data:
• Imax Maximal fold-gene induction of the luciferase gene over the full dose-response
up to 1000 μM
• EC 1.5, EC2, EC3 Concentration in μM for 1.5 / 2 / 3-fold gene induction
• Pos / Neg Rating of chemical according to prediction model
• reps. Positive number of independent repetitions positive / number of repetitions done
• IC50 Concentration in μM for 50% reduction of cell viability
• IC30 Concentration in μM for 30% reduction of cell viability

Positive control results:
Cinnamic aldehyde was run in all three repetitions and in all three masterplates used in the study. Chemicals 1 – 7 were run in masterplate1, 8 – 14 in masterplate 2, and 15 - 20 in masterplate 3.Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in all three
repetitions and in all three masterplates.
Key result
Run / experiment:
other: Luciferase induction
Parameter:
other: KeratinoSens
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: Cytotoxicity determinations
Parameter:
other: IC50
Remarks:
geom. mean
Value:
156.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: Luciferase determinations
Parameter:
other: Imax indicating maximal fold-induction up to a concentration of 1000 µM
Value:
1.25
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Prediction Model
Chemicals are rated positive if the following conditions are met (see also Scheme 1):
• The Imax indicates > 1.5-fold gene induction, and this induction is statistically significant above
the solvent control in a particular repetition as determined by students T-test. The EC1.5 value
is below 1000 μM in all three repetitions or in at least 2 repetitions. (If the Imax is exactly equal to
1.5, the chemical is still rated negative and no EC1.5 value is calculated by the evaluation
sheet.)
• At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC 1.5 determining
value), the cellular viability is above 70%.
• There is an apparent overall dose-response for luciferase induction, which is similar between
the repetitions.

Cytotoxicity determinations IC50 (ppm)
  Rep 1 Rep 2 Rep 3 Geometric mean
Test item 201.03 120.95 156.93 156.26

 Luciferase determinations  Imax         
   Rep1  Rep2  Rep3  Average Imax
 Test item  1.06  1.49  1.21

 1.25
Conclusions:
In summary, the test item revealed no sensitizing properties according to the KeratinoSens in vitro assay.
Executive summary:

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response.

The test chemical was dissolve in DMSO and tested according to the standard operating procedure of the KeratinoSens assay at 12 concentrations in three replicates. After 48 h incubation time, luciferase induction, and cellular viability at each concentration was determined. The compound had an IC50 value of 156.26 ppm, an Imax value of 1.25 and the EC value was < 1.5. So it can be concluded that the compound is not a skin sensitizer according to the KeratinoSens in vitro assay.

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

The skin sensitizing properties of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine were conducted in a guinea pig maximization test according to OECD 406 (Hüls AG, 1984). Twenty female guinea pigs were intradermally injected with a 0.1 % concentration of 2,2,4(or2,4,4)-trimethylhexane-1,6 -diamine (in water) and one week later epidermally exposed to a 10 % concentration of test substance. Twenty control animals were similary treated, but with vehicle alone. Two weeks after the epidermal application all animals were challegend with 2 % test substance and the vehicle.

Under the conditions of the test sensitizing effects were observed in 16 of the 20 animals at 24 and 48 hours. Based on these results, 2,2,4(or 2,4,4)-trimethylhexane-1,6 -diamine should be classified as contact sensitizer.

In a second guinea pig maximization test skin sensitizing properties of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine were conducted according to Magnusson and Kligman (1969). 15 guinea pigs were intradermally injected with a 0.5 % concentration of 2,2,4(or 2,4,4)-trimethylhexane-1,6 -diamine (in water) and epidermally exposed to a 0.5 % concentration of test substance. Control animals were similary treated, but with vehicle and Freunds Complete Adjuvant alone. Two weeks after the epidermal application all animals were challegend with 2 % test substance and the vehicle (Thorgeirsson, 1978).

Under the conditions of the test sensitizing effects were observed in 80 % of all animals at 24 hours. Based on these results, 2,2,4(or 2,4,4)-trimethylhexane-1,6 -diamine should be classified as contact sensitizer.


Migrated from Short description of key information:
2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine is skin sensitizing in two guinea pig maximization tests (Hüls AG, 1984 and Thorgeirsson, 1978).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Migrated from Short description of key information:
Assessment of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine concerning respiratory sensitisation is not possible because no data are available for this toxicological endpoint.

Justification for classification or non-classification

According to EC Regulation 1272/2008, classification is required as skin sensitising Cat.1 A; H317: "May cause an allergic skin reaction." Classification of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine concerning respiratory sensitisation is not possible because no data are available for this toxicological endpoint.