Triisooctylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The study was conducted due to provisions of other legislations than REACH.

Test material

Test material information

Constituent 1

Reference substance name:

Triisooctylamine

EC Number:

247-092-0

EC Name:

Triisooctylamine

Cas Number:

25549-16-0

Molecular formula:

C24H51N

IUPAC Name:

tris(6-methylheptyl)amine

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.: 0008924462

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: HARLAN (Kreuzelweg 53, 5961 NM HORST The Netherlands)
- Age at study initiation: 3 or 4 weeks old at the beginning of the main test
- Weight at study initiation: animals of comparable size and weight
- Housing: The animals were housed in groups of 2
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 30-70
- Air changes (per hr): at least 10 cycles per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

induction: 50 %

Challenge

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

challenge: 25 %

No. of animals per dose:

20 animals (treatment group)
10 animals (control)

Details on study design:

Preparation of test item
For the purpose of the study, the test item was prepared immediately prior to dosing in liquid paraffin for the topical applications (pretests). This vehicle was chosen as it produced the most suitable formulation at the required concentration. Indeed, the preparation of the test item at 50% in liquid paraffin (w/w) was a colorless solution.

Preparation of animals
Before the experimentation process, animals were identified individually by marking with picric acid and by means of a numbered ring on the edge of one ear.
The animals were carefully shorn before each test item application:
- On the inter-scapular zone for the induction phase,
- On the dorso-lumbar zone for the challenge phase.
At least 3 hours before the first reading (challenge phase) they were shorn a second time in this dorso-lumbar zone.
The animals were weighed at the beginning, before the second induction and at the end of the study.

Preliminary study
Maximum Non Irritant Concentration (M.N.I.C.) determination:
This test was carried out with a reduced number of animals, for the purpose of determining the maximal item concentration without risk of an irritant effect during the challenge phase.
Furthermore, this test evaluates the irritant potential of the test item, and defines, if possible, a mild to moderate irritant concentration during topical induction phase.
Four guinea pigs were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing (25mm x 50mm non woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for a period of 6 hours at 4 different concentrations: diluted at 50%, 25%, 10% and 5% in liquid paraffin.
Washing of the skin after removal of the dressing was done with liquid paraffin.
The animals treated at the concentrations of 50%, 25%, 10% and 5% diluted test item received 0.5 mL of the corresponding preparation.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings. The skin reaction was observed and recorded according to the grades described hereafter.
An additional guinea pig was treated with the test item placed onto the selected treatment site and covered with an occlusive dressing (25mm x 50mm non woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for a period of 6 hours at the concentration of 100%.
Washing of the skin after removal of the dressing was not necessary.
The animal treated at the concentration of 100% received 0.5 mL of the test item.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings. The skin reaction was observed and recorded according to the grades described hereafter.

Main study
GROUP 1 (negative control): 10 female guinea pigs
GROUP 2 (treated) : 20 female guinea pigs

Induction phase
After shearing of the scapular zone, the 3 local applications were performed on D0, D6 and D13 during 6 hours under occlusive dressing (25mm x 50mm non woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M).
The animals of the control group received 0.5 mL of liquid paraffin and the animals of the treated group received 0.5 mL of the test item diluted at 50% in liquid paraffin w/w.
Washing of the skin after removal of the dressing was not necessary.

Rest phase
The animals of both groups were left untreated for 13 days.

Challenge phase
The experimental procedure of this phase was identical for both groups Group 1 (Control) and Group 2 (Treated) according to this approach: on the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing (25mm x 50mm non woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M), was performed during 6 hours:
- 1 area containing 0.5 mL of the test item diluted at 25% in liquid paraffin w/w (MNIC = maximal non irritant concentration) on the left flank and one area containing 0.5 mL of liquid paraffin on the right flank.
Washing of the skin after removal of the dressing was not necessary.

Macroscopic examinations and evaluation of cutaneous reactions
A macroscopic evaluation of the cutaneous reactions (erythema and oedema) was conducted and all the local or systemic reactions were recorded as Group 1 (Control) and Group 2 (Treated):
• Approximately 24 hours after removal of the occlusive dressing, the cutaneous reactions were observed and graded according to the scales, given below.
• Approximately 24 hours later (i.e. 48 hours after removal of the occlusive dressing), a second observation was made.
• Approximately 24 hours later (i.e. 72 hours after removal of the occlusive dressing), a third observation was made during the main study.
Grading scale
0.......No visible change
1.......Discrete or patchy erythema
2.......Moderate and confluent erythema
3.......Intense erythema and swelling
All the animals with scores above or equal to 1 during the challenge phase, were considered positive.

Interpretation of reactions
The percentage of animals that showed a sensitivity contact potential is calculated 24 and 48 hours after the removal of the occlusive dressings.
A comparison of the intensities and persistence of reactions at the test item challenge sites in the test and control animals permits identification of sensitisation reactions.
=> If the test item at the maximum non-irritant concentration produces reactions in test group animals at the 24 or 48 -hour readings, these reactions were attributed to skin sensitisation. This pre-supposes that no similar reactions were observed in the test material challenge sites of any of the control group animals. If irritation was observed in the control group animals, only reactions in the test group animals that exceed the most severe reaction seen in the control group animals were attributed to skin sensitisation. The results were expressed in terms of incidence and severity of responses, ie:
Incidence Score: The number of test group animals showing skin reactions greater than the most severe reaction observed in the control group animals, expressed as a fraction of the total number of test group animals.
Severity Score: The sum of the values assigned to the skin responses at the test item challenge sites of the test and control group animals, at each evaluation, divided by the number of animals in that group.

Positive control substance(s):

yes

Remarks:

periodic positive control with α-Hexylcinnamaldehyde CAS n° 101-86-0

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

10

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

10

Results 3

Key result

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

10

Results 4

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %

No. with + reactions:

9

Total no. in group:

20

Results 5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

5

Total no. in group:

20

Results 6

Key result

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25 %

No. with + reactions:

4

Total no. in group:

20

Any other information on results incl. tables

RESULTS

Preliminary study

- MNIC determination:

24 hours after removal of the patches, a moderate erythema (grade 2) was noted in the animal treated at the concentration of 100% and a discrete erythema (grade 1) was noted in three animals (3/4) treated at the concentration of 50%. No cutaneous reaction was noted in the fourth animal at the concentration of 50%.

48 hours after removal of the patches, an intense erythema (grade 3) was noted in the animal treated at the concentration of 100% and a discrete erythema was noted in two animals (2/4) treated at the concentration of 50%.

24 and 48 hours after removal of the patches, no cutaneous reaction was noted at the concentrations of 25%, 10% and 5%

In view of these results, the concentration selected was 50% for the 3 inductions of the main study and the concentration selected was 25% (MNIC) for the challenge phase.

Main study:

- Induction phase

Moderate erythema (grade 2) was noted in twelve animals (12/20) and discrete erythema (grade 1) was noted in eight animals (8/20), after the first induction.

Moderate erythema was noted in nineteen animals (19/20) associated with dryness in seven animals (7/19), while discrete erythema was noted in one animal (1/20), after the second induction.

Intense erythema (grade 3) was noted in eighteen animals (18/20) associated with dryness in two animals (2/18), while moderate erythema was noted in two animals (2/20), after the third induction.

No cutaneous reaction was recorded during the induction phase in the control group.

- Challenge phase:

In the treated group (treatment dose of 25%), it was recorded a slight erythema (grade 1) in 45% (9/20), 25% (5/20) and 20% (4/20) of the animals, 24, 48 and 72 hours after the challenge phase, on the treated area, respectively.

In the control group (associated with the treatment dose of 25%), no cutaneous intolerance reaction was observed during the examination following the removal of the occlusive dressing.

No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (control item).

Weight evolution

No abnormalities and no differences in the body weight between the control and the treated group were observed.

Mortality

No mortality was registered during the main test.

Clinical signs

No abnormal clinical signs related to the administration of the test item were observed.

CONCLUSION

Under the conditions of this study, the test item Triisooctylamine was considered to be a skin sensitizer.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria