1,2-Propanediamine, N1-(2-aminoethyl)-3-(4-ethoxyphenyl)-, hydrochloride (1:2), (2S)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Guideline adopted 22 July 2010; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). The IMDS was validated and published with scientific justification in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

Deviations:

yes

Remarks:

- modification: in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance (Integrated Model for the Differentiation of Skin reactions (IMDS))

Principles of method if other than guideline:

This study is performed according to OECD TG 429. As stated in OECD TG 429 besides the classical radioactive method ‘other endpoints for assessment of the number of proliferating cells may be employed’ as so-called ‘me-too’ tests, if the required performance standards are fulfilled, they are ‘based on similar scientific principles and measure or predict the same biological or toxic effect’ and they are validated.
Here, an alternative method is used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes (IMDS LLNA; Integrated Model for the Differentiation of Skin Reactions). In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitizing properties. Information on validation of the IMDS LLNA and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
In the IMDS LLNA stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify skin sensitization) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
- Vohr, H.-W., Blümel, J., Blotz, A., Homey, B. and Ahr, H.J. An intra-laboratory validation of IMDS: Discrimination between (Photo) Allergic and (Photo) Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509 (2000).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(S)-1-(4-Ethoxybenzyl)-3-azapentane-1,5-diaminedihydrochloride

EC Number:

606-947-1

Cas Number:

221640-21-7

Molecular formula:

C13 H23 N3 O . 2 Cl H

IUPAC Name:

(S)-1-(4-Ethoxybenzyl)-3-azapentane-1,5-diaminedihydrochloride

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: harlan Nederland, 5960 AD Horst, The Netherland
- Mean weight at study initiation: 26 - 36 g
- Housing: 8 animals per cage during adaption period; 1 animal per cage during the study
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- rel. Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 2, 10, 30 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- body weights at the start and the end of the study

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control group by the Mann-Whitney or the Wilcoxon signigicance test (Rank Sum Test or One Way ANOVA or Kruskal-Wallis ANOVA) at significance levels of 5 % (one-tailed for LLNA/IMDS or PNLA (larger)). Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differentes in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.97

Test group / Remarks:

positive control

Results 2

Parameter:

SI

Value:

1.2

Test group / Remarks:

30% treatment

Results 3

Parameter:

SI

Value:

1.21

Test group / Remarks:

10% treatment

Results 4

Parameter:

SI

Value:

0.88

Test group / Remarks:

2% treatment

Results 5

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control

Results 6

Parameter:

SI

Remarks on result:

other: No significant dose-dependent increase in the stimulation indices for the weight or cell counts as well as for ear swelling or ear weights.

Any other information on results incl. tables

Table 1: Summary of the LLNA/IMDS results (means of 6 animals per group)

Parameter investigated	Vehicle control	Dose 2 %	Dose 10 %	Dose 30 %
Stimulation index: weight of draining lymph nodes	1.00	0.89	1.25	1.11
Stimulation index: cell count in draining lymph nodes	1.00	0.88	1.21	1.20
Ear swelling in 0.01 mm on day 4 (index)	18.25 (1.00)	18.42 (1.01)	18.25 (1.00)	18.08 (0.99)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	14.01 (1.00)	13.84 (0.99)	13.63 (0.97)	13.34* (0.95)

*statistically significant increase (p <= 0.05)

The test item has no sensitizing potential in mice after dermal application of up to and including a 30 % concentration. No indication for a non-specific (irritant) activation was detected either. Therefore, the concentration of 30 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization. These findings were verified by the results obtained with the positive control compound Alpha Hexyl Cinnamic Aldehyde.

The body weights of the animals were not affected by the treatment.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 2, 10 and 30 % formulated in dimethylformamide were tested. The results show that the test item has neither a non-specific (irritant) nor a specific immunostimulating (sensitizing) potential.These findings were verified by the results obtained with the positive control.

Executive summary:

In a dermal sensitization study according to OECD TG 429 (24 April 2002) with Triamine Dihydrochloride in Dimethylformamide, young adult female NMRI Mice (6/dose) were tested in the LLNA (OECD TG 429). Up to 30% of the test item was applied. Hexyl cinnamic aldehyde (CAS No 101-86-0) was used as positive control substance. Only after treatment with the positive control, alpha hexyl cinnamic aldehyde, the NMRI mice showed statistically significant increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to vehicle control animals.

This study did neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the test substance. In this study, the test item is not a dermal sensitizer.