Triisooctylamine

Endpoint summary

Description of key information

LLNA: sensitizer
Buehler-Test: sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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.

Specific details on test material used for the study:

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

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

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

induction: 50 %

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

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

10

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

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %

No. with + reactions:

9

Total no. in group:

20

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

5

Total no. in group:

20

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

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.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

LLNA

In this study the test item Triisooctylamine was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA, OECD 429 guideline and GLP) in mice. Test item solution at different concentrations was prepared in the vehicle MEK. The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations. For this purpose a local lymph node assay was performed using test item concentrations of 0.25, 0.5, and 1% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by four pre-experiments). The animals showed neither any signs of systemic toxicity nor local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant increase in ear weights was observed in the mid and the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. The mid and high dose group reached or slightly exceeded this threshold (indices of 1.1 and 1.17, respectively). However, this was considered to be not biologically relevant, as the observed increase did not exceed the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429. Nevertheless, the increased ear weight values indicated an irritant property of the test substance.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.24, 3.13 and 16.62 were determined with the test item at concentrations of 0.25, 0.5, and 1% (w/w) in MEK, respectively. A clear dose response was observed. An outlier was identified in the high dose group (DPM value determined for animal number 18). However, as exclusion of the outlier did not change the overall test result, the value in question was not excluded from calculation. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and - cell count was observed in the mid and high dose groups in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in the mid and high dose groups (indices of 1.9 and 4.6, respectively).

Based on the above mentioned findings regarding ear skin irritation, an influence of irritation on lymphocyte proliferation cannot be excluded and thus a guinea pig test according to OECD 406 guideline could be helpful to elucidate whether the observed statistically significant and biologically relevant increase in DPM value and also in lymph node weight and - cell count in the mid and high dose groups was partially or completely caused by the observed ear skin irritation. Nevertheless, on the basis of the present data, the test item has to be classified as a sensitiser. Thus, the test item Triisooctylamine was found to be a skin sensitizer and an EC3 value of 0.48 % was derived.

GPMT

The aim of the study was to re-evaluate the possible allergenic activity of the test item Triisooctylamine after topical administration in guinea pigs and to elucidate whether skin irritation partially or completely contributed to the skin sensitization observed in the LLNA reported above.

After induction of 20 guinea-pigs by 3 topical applications with the test item Triisooctylamine applied as 50% solution in liquid paraffin under occlusive dressing and a 14-day rest phase, the challenge phase, under occlusive dressing for 6 hours, consisted of a single topical application of the test item diluted at 25% in liquid paraffin and of a negative control (liquid paraffin). The experimental protocol was established from the O.E.C.D. Test Guideline No.406 dated July 17th, 1992, and the method B.6 of the Council regulation No. 440/2008 and the US EPA OPPTS guideline 870.2600 (March 2003).

In the treated group (treatment dose of 25%), it was recorded a slight erythema 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).

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

Conclusion:

Based on these results in the guinea pig test it can be concluded that skin irritation must have partially contributed to or must have intensified the skin sensitization reaction observed in the LLNA as the results of both tests are giving different potency information. According to the results of the LLNA, the test substance would be of high potency and would have to be classified as Cat. 1A skin sensitizer. Based on the results of the Buehler test, the substance is only of low to moderate potency and would require a classification as Cat. 1B skin sensitizer. In light of these conflicting results, the substance is classified as Cat. 1 skin sensitizer without applying a sub-categorization.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified as skin sensitizer cat 1 without subcategoriization (H317 "May cause an allergic skin reaction") under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.