L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

ethyl (2S)-3-[3,5-diamino-4-(4-methoxyphenoxy)phenyl]-2-acetamidopropanoate

Cas Number:

440667-78-7

Molecular formula:

C20H25N3O5

IUPAC Name:

ethyl (2S)-3-[3,5-diamino-4-(4-methoxyphenoxy)phenyl]-2-acetamidopropanoate

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species: Mice CBA/CaOlaHsd
Source: Anlab, Praha, Czech Republic
Number and Sex of Animals: 28 females
Age at First Dose: 9-10 weeks; female animals were non-pregnant and nulliparous
Animal Health: The health condition of animals and skin integrity was examined by a veterinarian before initiation of the study. Animals were healthy, without visible symptoms of disease.
Acclimation: The animals were acclimated to the conditions identical to the condition during the experiment 5 days prior to the start of treatment. The acclimation was performed according to standard operation procedure ŠPP/EZ V001.
Housing Condition: The animals were housed in TECNIPLAST cages from the Tecniplast Company, in Conventional animal room No: B2-608. 5 females were housed per cage. The room temperature was within the range of 20-24oC, relative humidity was within the range of 40-60%. The animals were subjected to a 12-hour light/ 12-hour dark cycle. The sanitation was performed according to SOP ŠPP/EZ/V005.
Diet : A laboratory food for mice V1534-000 R/M-H (Ssniff) was served ad libitum. The certificate of analysis is included in the raw data.
Water: The animals received tap water for human consumption ad libitum. The water from the local mains was monitored for quality by testing for the microbiological and chemical quality by Waterworks Bratislava quarterly. Bottles were exchanged and cleaned once a week. The quality of drinking water is periodical monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.
Bedding: Sterilized bedding from JRS Lignocel®. Hygienic animal bedding, sterilized sawdust.
The cages were cleaned once a week. The cages were emptied and cleaned with water and detergent. After cleaning they were dried and then immersed in disinfectant. The cage racks were cleaned in the rooms every week manually with water and detergent.
Animals Identification: Each animal was marked by code on the tail base in accordance with SOP: ŠPP/TOX/V002 to identify the animal individually. Each cage was marked with a coloured cage card. Each cage was affixed with a cage card containing pertinent animal and study information.
Justification for the Choice of Species: The CBA/Ca mice are the standard experimental rodent of choice and recommended by OECD Guideline No. 429.
Randomisation: Before the treatment animals were randomized based on body weights to treatment groups.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

The doses were selected from the concentration series 100 %, 50 %, 25 %, 10 %, 5 %, 2.5 % etc. according to OECD Guideline No. 429.
The starting concentration was determined according to Pre-screen test results.

Number of animals:
Pre-screen test:
Group Group name No. animals ID number Cage number
1 L-TDOM 100 % (w/v) 1 1 1
2 L-TDOM 50 % (w/v) 1 1 2
3 L-TDOM 25 % (w/v) 1 1 3
Legend: L-TDOM - L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester


Main study:
Group Group name No. animals ID number Cage number
1 Negative Control 5 1-5 1
2 Positive Control 5 6-10 2
3 L-TDOM 10 % (w/v) 5 11-15 3
4 L-TDOM 25 % (w/v) 5 16-20 4
5 L-TDOM 50 % (w/v) 5 21-25 5
Legend: L-TDOM - L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester

No. of animals per dose:

5

Details on study design:

Day 1:
Each animal was identified, and the body weight was recorded. To the dorsum of each ear 25 µL of the appropriate dilution of the test item, positive control or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5:
No treatment.
Day 6:
The body weight of each animal was recorded. 250 μL of sterile phosphate-buffered saline (PBS) containing 20 μCi (7.4×105 Bq) of tritiated (3H)-methyl thymidine was injected into all test and control mice via the tail vein.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear was excised and pooled in PBS for each experimental group (pooled treatment group approach).

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.96

Test group / Remarks:

50% group

Results 2

Key result

Parameter:

SI

Value:

0.94

Test group / Remarks:

25% group

Results 3

Key result

Parameter:

SI

Value:

0.78

Test group / Remarks:

10% group

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitization potential of L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester
was evaluated by LLNA method, which basic underlying principle is that sensitizers induce a primary proliferation of lymphocytes in the auricular lymph nodes draining the site of chemical application.
In the present study, the test item was applied to the dorsum of each ear of five female mice (CBA/CaOlaHsd) per group over three consecutive days, at three concentrations (10 %, 25 % and 50 % w/v). All animals survived throughout the test period without showing any clinical signs of toxicity. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance as a sensitizer. Therefore, it was not possible to calculate an EC3 value.
These results demonstrate that the test item L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester was not a skin sensitizer under the test conditions of this study

Executive summary:

The skin sensitizing potential of L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester was assessed using the murine local lymph node assay. Based on the results of this study, L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester is not considered a skin sensitizer under the conditions of this LLNA study.