Carbamic acid, [(1S)-2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-, phenylmethyl ester, dihydrochloride

Endpoint summary

Description of key information

Skin sensitisation (LLNA, mouse): not sensitising [Leidenfrost 2014]

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

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
- Age at study initiation: 6 weeks
- 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

Vehicle:

dimethylformamide

Concentration:

0, 2, 10, 30 %

No. of animals per dose:

6

Details on study design:

PRE-SCREEN TESTS: no specific preliminary test was performed
- Irritation: The test item showed irritating effects to the skin and is classified as corrosive to the eye (20% [w/v])

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT: Six animals were placed in each group.
- Criteria used to consider a positive response:
-Increase of the SI (cell count) in the draining lymph nodes above 1.4,
- Differentiation indices (DI), which is the quotient of the relative lymph node reaction divided by the relative acute skin reaction should be > 1
- The EC 1.4 value > 3,
- Exceeding the ear swelling positive level of 10%

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.

Key result

Parameter:

SI

Test group / Remarks:

positive control (alpha hexyl cinnamic aldehyde) 2.5%, 10%, and 40%

Remarks on result:

other: A check is done in regular intervals using adequate vehicles. The last reliability test using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentrations of 2.5, 10 and 40% showed clear positive results

Key result

Parameter:

SI

Value:

2.49

Test group / Remarks:

30% treatment group

Key result

Parameter:

SI

Value:

2.01

Test group / Remarks:

10% treatment group

Key result

Parameter:

SI

Value:

1.27

Test group / Remarks:

2% treatment group

Remarks on result:

other:

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control

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	1.21	1.74*	2.11*
Stimulation index: cell count in draining lymph nodes	1.00	1.27*	2.01*	2.49*
Ear swelling in 0.01 mm on day 4 (index)	17.75 (1.00)	18.08 (1.02)	18.67 (1.05)	19.92* (1.12)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	12.69 (1.00)	12.75 (1.00)	14.50* (1.14)	17.82* (1.40)

*statistically significant increase (p <= 0.05)

After treatment with test substance there were clear increases compared to control animals regarding the weights and the cell counts of the draining lymph nodes, which are of statistical significance in the mid and high dose group. The "positive level", which is 1.4 for cell counts, has been exceeded in these dose groups. A sensitizing potential can be assumed from the increase in cell proliferation in the draining lymph nodes.The EC 1.4 value calculated is 3.4 % for this test item. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC (2003) this value corresponds to a moderate skin sensitizer. The ¿positive level¿ of ear swelling which is 2 x 10-2 mm increase, i.e. more than 10 % increase, has been exceeded in the high dose group. A significant increase compared to vehicle treated animals regarding ear weight was detected in the mid and high dose group. From these data it may be concluded that the test item has an irritant potential at the highest concentration tested, i.e. 30 % in addition to its sensitizing potential..

It has to be clarified that the "positive levels" mentioned above are exclusively defined for the NMRI outbred mice used for this study. Such positive limits have to be calculated for each strain of mice individually.

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

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

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 a non-specific (irritant) and a specific immunostimulating (sensitizing) potential. Based on the data of this study the test substance has to be classified as a moderate skin sensitizer. Therefore, the concentration of 2% turned to be the NOEL for the parameters investigated in this study. These findings were verified by the results obtained with the positive control.

Executive summary:

In a local lymph node assay according to OECD guideline 429 (2010) Z-Triamine Dihydrochloride dissolved in dimethylformamide was assessed for its possible contact allergenic potential using test item concentrations of 2, 10 and 30%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.27, 2.01, and 2.49 were determined with the test item at concentrations of 2, 10, and 30% in dimethylformamide, respectively. These results indicate that the test substance could elicit an SI ≥ 1.4 (according to the modified classification criteria used in this study) and Z-Triamine Dihydrochloride is therefore regarded as skin sensitiser under the conditions of this study. The data showed a dose-response and an EC1.4 value > 10% was calculated.

Based on these results Z-Triamine Dihydrochloride is classified as Category 1B based on CLP, EU GHS (Regulation (EC) No 1272/2008).

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In a local lymph node assay according to OECD guideline 429 (2010) Z-Triamine Dihydrochloride dissolved in dimethylformamide was assessed for its possible contact allergenic potential using test item concentrations of 2, 10 and 30%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.27, 2.01, and 2.49 were determined with the test item at concentrations of 2, 10, and 30% in dimethylformamide, respectively. These results indicate that the test substance could elicit an SI ≥ 1.4 (according to the modified classification criteria used in this study) and Z-Triamine Dihydrochloride is therefore regarded as skin sensitiser under the conditions of this study. The data showed a dose-response and an EC1.4 value > 10% was calculated.

Based on these results Z-Triamine Dihydrochloride is classified as Category 1B based on CLP, EU GHS (Regulation (EC) No 1272/2008).

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation:

mouse local lymph node assay (OECD TG 429): positive

According to Regulation (EC) No 1272/2008, Annex I Z-Triamin-Dihydrochlorid shall be allocated to Category 1B (H317)

Respiratory sensitisation:

no data available