Amines, N'-[3-[(3-aminopropyl)amino]propyl]-N,N-di-C16-18-alkyltrimethylenedi-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September 2016 - 10 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification in vivo testing:
An in vivo study for the evaluation of skin sensitisation was needed for an on-going notification outside of EU, where in vivo results were demanded.
Further, the substance is not suitable for testing in the currently acceptable in vitro models:
- DPRA: A test chemical should be soluble in an appropriate solvent at a final concentration of 100 mM. However, test chemicals that are not soluble at this concentration may still be tested at lower soluble concentrations and in such a case positive results could be used to identify a test chemical as sensitiser. In case of negative prediction (lack of reactivity) no firm conclusion should be drawn.
Di-C16-C18 (evennumbered) alkyl tripropylenetetramine is an UVCB, and no definable molecular weight makes it not suitable for DPRA; besides, the estimated solubility is 30 µg/L or lower, whereas required solubility 100 mM ~70 g/L. Therefore, DPRA cannot results to a firm conclusion.
- KeratinoSens: The test method is applicable to test chemicals that are soluble or that form a stable dispersion either in water or DMSO. The highest concentration required in the test method is 2000 μM. However, if the highest concentration of 2 000 μM cannot be obtained e.g. due to limited solubility or cytotoxic properties of the test chemical, lower concentrations can be used. Negative results obtained with concentrations < 1 000 μM should be considered as inconclusive.
For Di-C16-C18 (evennumbered) alkyl tripropylenetetramine, 1000 µM would be about 0.7 g/L. With an estimated solubility of 30 µg/L, this test will not lead to conclusive resulst.
- h-CLAT: Applicable to test chemicals soluble or that form a stable dispersion in an appropriate solvent.
Substances with Log Kow up to 3.5 can be tested whereas substances with Log Kow higher than 3.5 tend to produce negative results. For such substances positive results could be used to support the identification of a test chemical as sensitiser. Negative results should be considered as inconclusive.
Di-C16-C18 (evennumbered) alkyl tripropylenetetramine has an logPow ≥ 13, and thus inconclusive is best result to be expected.

Justification GPMT testing:
The substance is highly irritating to skin; the mechanism for this skin reaction is likely inflammatory mediated (Considering skin responses in vivo are not supported in RhE systems as Episkin). For this sort of substances the LLNA is not suitable and is expected to lead to false-positive results.
In recently published articles in peer reviewed journals, see reference list, it is clearly demonstrated that irritants and surfactants are more likely to give rise to false positives in the LLNA.
Consequently, in the evaluation of such substances for sensitizing properties the LLNA test is not an appropriate assay and would not represent an optimum use of test animals. It is therefore recommended that the Guinea Pig Maximisation Test (GPMT) is used instead. This is also supported by the TG OECD 406 "In addition, test substance classes or substances containing functional groups shown to act as potential con founders (Basketter et al., 2009) may necessitate the use of guinea pig tests".

References:
1. Kreiling R, Hollnagel HM, Hareng L, Eigler D, Lee MS, Griem P, Dreessen B, Kleber M, Albrecht A, Garcia C, Wendel A. (2008).
Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT).
Food Chem. Toxicol. 46. 1896-1904.
2. David Basketter, Nicholas Ball, Stuart Cagen, Juan-Carlos Carrillo, Hans Certa, Dorothea Eigler, Christine Garcia, Harald Esch, Cynthia Graham, Carl Haux, Reinhard Kreiling, Annette Mehling. (2009)
Application of a weight of evidence approach to assessing discordant sensitisation datasets: Implications for REACH
Regul Toxicol Pharmacol. 55: 90–96
3. Garcia C, Ball N, Cagen S, Carrillo JC, Certa H, Eigler D, Esch H, Graham C, Haux C, Kreiling R, Mehling A. (2010)
Comparative testing for the identification of skin-sensitizing potentials of nonionic sugar lipid surfactants.
Regul Toxicol Pharmacol. 58(2):301-307.
4. Nicholas Ball, Stuart Cagen, Juan-Carlos Carrillo, Hans Certa, Dorothea Eigler, Roger Emter, Frank Faulhammer, Christine Garcia, Cynthia Graham, Carl Haux, Susanne N. Kolle, Reinhard Kreiling, Andreas Natsch, Annette Mehling. (2011)
Evaluating the sensitization potential of surfactants: Integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach
Regul Toxicol Pharmacol. 60: 389–400

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

May 2008, including most recent amendments

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147.

Version / remarks:

November 2000; including the most recent partial revisions

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Maximization test was selected since in recently published articles in peer reviewed journals (Kreiling R. et al., 2008; Basketter D. et al., 2009; Garcia C. et al., 2010; Ball N. et al., 2011), it is clearly demonstrated that irritants and surfactants are more likely to give rise to false positives in the LLNA. Consequently, in the evaluation of such substances for sensitizing properties the LLNA test is not an appropriate assay and would not represent an optimum use of test animals. As the di-C16-C18 (evennumbered) alkyl tripropylenetetramine is a surfactant and irritating to the skin, it is therefore recommended that the Guinea Pig Maximisation Test (GPMT) is used instead. This is also supported by the TG OECD 406 "In addition, test substance classes or substances containing functional groups shown to act as potential con founders (Basketter et al., 2009) may necessitate the use of guinea pig tests".

Test material

Test material information

Constituent 1

Reference substance name:

[3-({3-[(3-aminopropyl)amino]propyl}amino)propyl](hexadecyl)octadecylamine; [3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dihexadecylamine; [3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dioctadecylamine

EC Number:

941-593-4

Cas Number:

1623405-26-4

Molecular formula:

Not applicable UVCB

IUPAC Name:

[3-({3-[(3-aminopropyl)amino]propyl}amino)propyl](hexadecyl)octadecylamine; [3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dihexadecylamine; [3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dioctadecylamine

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): di-C16-C18 (evennumbered) alkyl tripropylenetetramine
- Purity: 100% (UVCB substance)
- Purity test date: 08 July 2016
- Batch No.: 1330539
- Expiration date of the batch: 04 August 2018
- Appearance: Viscous liquid
- Storage condition of test material: At room temperature container flushed with nitrogen
- pH: 8.5-10.5 at concentration of 1%
- Specific gravity/density: 0.864 at 20°C

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’arbresle, France.
- Age at study initiation: Young adult animals (approx. 7 weeks old)
- Housing: Group housing of maximally 5 animals per labeled cage.
- Diet (e.g. ad libitum): Complete breeding diet for guinea pigs (SSNIFF® MS-H, SSNIFF® Spezialdiäten GmbH, Soest, Germany). Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

0.2%

Day(s)/duration:

1

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

5%, 0.5 mL

Day(s)/duration:

day 8, for 48 hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

2%

Day(s)/duration:

24 h

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

2%

Day(s)/duration:

24h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

2*10 per test group
Control 2*5 per group

Details on study design:

RANGE FINDING TESTS: (8 animals)
Series of test item concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 5%, 2%, 1%, 0.5, 0.2%, 0.1%, 0.05%, 0.02%.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The six animals selected were 4 weeks old. No body weights were determined.

Intradermal injections:
Initially, a series of four test item concentrations was tested; the highest concentration was the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hours after treatment.
Based on the results in the initially treated animals, four additional animals were treated in a similar manner at a later stage since the results were inconclusive and no dose response was seen. This did not affect the concentration selection for the main study.

Epidermal application:
A series of four test item concentrations was tested; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape#, which were held in place with Micropore tape and subsequently Coban elastic bandage#. The initially used animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test item using water.
The resulting dermal reactions were assessed for irritation 24 and 48 hours after removal of the dressings. Based on the results in the initially treated animals, four additional animals were treated in a similar manner at a later stage since the results were inconclusive and no dose response was seen. This did not affect the concentration selection for the main study.

MAIN STUDY
INDUCTION EXPOSURE
- No. of exposures: 1
Day 1 The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Sigma-Aldrich, Steinheim, Germany) with water for injection (B.Braun Melsungen AG, Melsungen. Germany).
B) The test item at a 0.2% concentration.
C) A 1:1 w/w mixture of the test item, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).
Day 3 The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 8 The scapular area between the injection sites was clipped and subsequently treated with 0.5 mL of a 5% test item concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test item using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

INDUCTION - CONTROL ANIMALS
The control animals were treated as described for the experimental animals except that, instead of the test item, the vehicle was administered.

CHALLENGE - ALL ANIMALS
Day 21 One flank of all animals was clipped and treated by epidermal application of a 2% test item concentration and the vehicle (0.1 mL each), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 24 hours exposure and the skin cleaned of residual test item and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
Rechallenge – Additional animals
Day 28 A re-challenge was conducted approximately one week after the first challenge, to clarify the results in the first challenge. The contralateral flank of all animals was similarly treated.
After termination, animals were sacrificed using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and an intra-peritoneal injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Challenge controls:

Not applicable

Positive control substance(s):

yes

Remarks:

the results of the latest reliability check, performed in April 2016 with Alpha- Hexylcinnam aldehyde, are reported

Results and discussion

Positive control results:

The latest reliability check (performed less than 6 months ago) resulted to a sensitisation rate of 33 percent to a 20% concentration of alpha-Hexylcinnamaldehyde.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2%

No. with + reactions:

9

Total no. in group:

20

Remarks on result:

other: See comments to results

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2%

No. with + reactions:

4

Total no. in group:

20

Remarks on result:

other: See comment to results

Results 3

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

2%

No. with + reactions:

5

Total no. in group:

10

Remarks on result:

other: See comments to results

Results 4

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

2%

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

other: See comments to results

Results 5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

2%

No. with + reactions:

0

Total no. in group:

10

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

2%

No. with + reactions:

0

Total no. in group:

10

Results 7

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

2%

No. with + reactions:

0

Total no. in group:

5

Results 8

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

2%

No. with + reactions:

0

Total no. in group:

5

Results 9

Key result

Reading:

other: Overall

Hours after challenge:

24

Group:

positive control

Dose level:

20% Alpha- Hexylcinnamaldehyde, technical grade

No. with + reactions:

3

Total no. in group:

9

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

 

First challenge reading

Animal number	24 h		48 h
	Test item concentration 2%	Vehicle	Test item concentration 2%	Vehicle
Control
11	0	0	0S	0
12	0	0	0S	0
13	0	0	0	0
14	0	0	0	0
15	0	0	0	0
Experimental
16	0S	0	0S	0
17	1	0	0S	0
18	0S	0	0S	0
19	1	0	1S	0
20	0	0	0S	0
21	0	0	0S	0
22	0	0	0S	0
23	1	0	1KS	0
24	0	0	0S	0
25	1	0	0S	0

 

Second challenge reading in second group of ten test and 5 control animals:

Animal number	Day 23		Day 24
	Test item concentration 2%	Vehicle	Test item concentration 2%	Vehicle
Control
26	0	0	0	0
27	0	0	0S	0
28	0	0	0	0
29	0	0	0S	0
30	0	0	0	0
Experimental
31	1	0	1S	0
32	0S	0	0S	0
33	1S	0	0S	0
34	1	0	1KS	0
35	0S	0	0S	0
36	1	0	0S	0
37	0	0	0S	0
38	1	0	1KS	0
39	0	0	0	0
40	0	0	0S	0

 

Rechallenge second group:

Animal number	Day 30		Day 31
	Test item concentration 2%	Vehicle	Test item concentration 2%	Vehicle
Control
26	0	0	0	0
27	0	0	0	0
28	0	0	0	0
29	0	0	0	0
30	0	0	0	0
Experimental
31	1	0	0S	0
32	0	0	0	0
33	1	0	0S	0
34	1K	0	1K	0
35	0	0	0S	0
36	1	0	0S	0
37	0	0	0S	0
38	1	0	1	0
39	0	0	0	0
40	0	0	0S	0

 

K. Scabs

S. Scaliness

Grading challenge reactions:

0. No visible change

1. Discrete or patch erythema

 

Histopathology results

 

SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GROUP/SEX

Necropsy Status: TERMINAL SACRIFICE GROUP (K0)

Incidence table - Selected findings with grades

Sex	Females
Dose Group	Control	Test
No. Animals per Dose Group	5	10
SKIN TEST SITE (A)           No.Examined	5	10
- Hyperplasia epidermal
GRADE 1	2	6
GRADE 2	-	1
TOTAL AFFECTED	2	7
MEAN GRADE/TISS.EXAMINED	0.4	0.8
- Inflammatory cell infiltrate
GRADE 1	2	8
GRADE 2	1	2
TOTAL AFFECTED	3	10
MEAN GRADE/TISS.EXAMINED	0.8	1.2
- Edema dermal
GRADE 2	-	1
TOTAL AFFECTED	-	1
MEAN GRADE/TISS.EXAMINED	-	0.2
SKIN CONTROL SITE (B)           No.Examined	5	10
- Inflammatory cell infiltrate
GRADE 1	-	3
TOTAL AFFECTED	-	3
MEAN GRADE/TISS.EXAMINED	-	0.3

 

Control animals: Individual findings and grading

Animal number	26	27	28	29	30
SKIN TEST SITE (A)
- Hyperplasia epidermal	-	1	-	-	1
- Inflamm.dermal	-	2	1	-	1
SKIN CONTROL SITE (B)	-	-	-	-	-

 

Test animals: Individual findings and grading

Animal number	31	32	33	34	35	36	37	38	39	40
SKIN TEST SITE (A)
- Hyperplasia epidermal	-	-	1	2	1	1	-	1	1	1
- Inflamm.dermal	1	1	1	2	1	1	1	2	1	1
- Edema dermal	-	-	-	2	-	-	-	-	-	-
SKIN CONTROL SITE (B)
- Inflamm.dermal	-	-	-	-	1	-	-	1	1	-

 

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The resulst of this GPMT study do not indicate that thsi substance is a skin sensitiser.

Executive summary:

Hypersensitivity in the Albino Guinea Pig (Maximization Test) according to OECD guidelines no. 406 (1992) "Skin Sensitization".

The study was based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens" (1970).

Test item concentrations selected for the main study were based on the results of a preliminary study.

In the main study, ten experimental animals were intradermally injected with a 0.2% concentration and epidermally exposed to a 5% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). 

Two weeks after the epidermal application all animals were epidermally challenged with a 2% test item concentration and the vehicle. Since it could not be decided whether the item is a sensitizer or not, additional animals were treated similarly. One week after the first challenge the additional animals were rechallenged using the same procedures.

When the results of the two first challenges are combined this results in a discrete or patchy erythema in 45% of the animals at 24 hours after exposure and which was still observed in 25% of the animals at 48 hours after exposure when compared to the control animals. In the rechallenge similar responses of 50% and 20% were found, at 24 and 48 hours after exposure respectively. No skin reactions other than scaliness were evident in all control animals.

Histopathology of the rechallenge of the additional animals showed epidermal hyperplasia (70%), inflammatory cell infiltrate (100%), and edema (10%) in the experimental animals treated with test item. However, inflammatory cell infiltrate was also found in 30% of the experimental animals challenged with the control substance.

In the control animals 40% showed epidermal hyperplasia and 60% showed inflammatory cell infiltrate. No abnormalities were shown when the control animals were treated with the control substance.

 

Considering that the skin reactions from the challenge with 2% were also observed in some animals during preliminary irritation testing, that none of the reactions attain a grade which should be regarded as indicative of skin sensitisation, and were already for a large part resolved at the second reading after 48 hours, it is most likely that the observed reactions are associated with irritation and local toxicity rather than an indication to sensistisation.

Also histological examination indicated that reactions in the test animals did not reach levels above what was already observed in control animals.

 

The results are therefore not considered to indicate potential sensitisation.