Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
May 2008, including most recent amendments
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Qualifier:
according to
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. certificate)
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

Reference
Name:
Unnamed
Type:
Constituent
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)

Inductionopen allclose all
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
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
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
2%
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
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)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2%
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
other: See comments to results
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
2%
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
other: See comment to results
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
2%
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
other: See comments to results
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: See comments to results
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
2%
No. with + reactions:
0
Total no. in group:
5
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.